Jarrell Miller will not face Anthony Joshua on June 1 after promoter Eddie Hearn reported a second "adverse finding" from a drugs test.
Miller's participation for the New York heavyweight bout was already under threat after the Voluntary Anti-Doping Association (VADA) informed both camps of an adverse finding in a drug-testing sample, leading to him being denied a licence for the date at Madison Square Garden.
Miller retained the right to request a further sample and to reapply for that licence to challenge Joshua in what will be the champion's US debut.
However, Hearn now says he has learned of a second alleged doping violation by Miller, meaning that fight is now dead in the water.
It was on March 20 when there was an adverse finding for GW1516, a metabolic modulator also known as cardarine and endurobol.
Hearn had already started the search for alternative opponents from the point of VADA's first update, and he wrote on social media on Friday: "We have now received news from VADA that Jarrell Miller has now failed a second separate test for a further substance.
"AJ's June 1 opponent will be announced next week. If you don't think you can beat him clean then delete my number!"
Hearn's Matchroom Sport had long planned to build Joshua's profile in the US boxing market, to the extent that the fight location, which they remain committed to, and Brooklyn's Miller had been selected specifically to maximise its appeal.
Responding to the initial adverse finding on Tuesday, Miller had written on Instagram on Thursday: "I have never knowingly taken any banned substance and when I found out the news I was totally shocked.
"My team and I stand for integrity, decency and honesty and together we will stand to fight this with everything we have.
"This was a voluntarily test that I was very happy to do and these results came just one week after another voluntarily test that I had taken which was completely clean."
Press Association Sport understands several opponents, including fellow American Michael Hunter, remain in contention to replace Miller.
Attempts were made to contact representatives of both Miller and VADA, but neither were available for comment.
Press Association Sport explores six potential opponents for Anthony Joshua.
Poland's undefeated Kownacki is based in New York and therefore has a suitable profile for the occasion of Joshua's United States debut, which is specifically why Miller was first selected. He would also earn a career-high purse by agreeing to the relatively late-notice fight.
A rematch between Whyte and Joshua has long appeared inevitable, to the extent that he had been in contention to fight his bitter rival at Wembley Stadium earlier this year. A potential stumbling block, amid his low profile in the US, is the fact Matchroom are preparing for him to be involved in a pay-per-view fight in London in July.
Given Hunter is American and has fought and won in the UK, his profile may yet be considered suitable for an occasion Matchroom hope will further Joshua's profile both domestically and in the US. That he is already signed to Joshua's promoters is a further positive, as is his present place on a bill they are promoting in Chicago the weekend before June 1, which means he should already be in training. His only defeat came against the great Ukrainian, Oleksandr Usyk.
Cuba's cultured Ortiz is still widely considered the division's dangerman; he perhaps remains the biggest threat to Joshua, WBC champion Deontay Wilder and Tyson Fury, and would represent a far lower reward opponent. The 40-year-old almost stopped the undefeated Wilder when they fought in New York last year.
The Germany-based Syrian was previously denied a high-profile fight with David Haye when, in 2013, the Brit withdrew having suffered a hand injury. His career has since stalled, and at 34 a fight with Joshua would provide an unexpected, lucrative opportunity. In 2016 he went the distance with Sefer Seferi, Fury's unremarkable comeback opponent, which demonstrates his decline. He also last fought in November 2017.
The undefeated German and European heavyweight champion is known in Britain for defeating Dereck Chisora in 2017, but is yet to fight beyond European level, making a bout against one of the world's two most dangerous heavyweights a significant risk. He also has minimal appeal in the US, having never fought outside of Europe, so appears an unlikely alternative to Miller.