Andy Murray's winning run at the Rafa Nadal Open was halted by world number 240 Matteo Viola, who won 3-6 6-4 7-6.
Murray, competing in an ATP Challenger Tour event for the first time since 2005, had secured back-to-back wins in Mallorca as he continues his comeback from hip surgery, but came unstuck against an inspired opponent.
The 32-year-old Scot struggled to maintain his performance levels after breaking Viola's serve twice to win the first set in 51 minutes.
The former world number one never recovered after falling 3-0 behind in the second, and although he fought back from 2-0 down after losing his opening service game in the final set, he did not produce his best.
Both players held serve from 2-2 to force a tie-break, which Viola went on to dominate, winning the first three points and going 6-2 up.
Murray hit a brilliant forehand return on Viola's first match point but served a double fault on his second to hand the Italian the match.
Murray had completed straight-set wins over teenager Imran Sibille and Norbert Gombos in the previous rounds but did not look comfortable against Viola, whose highest career singles ranking is 118.
Three-time major winner Murray will consider his defeat a setback as he builds up his match fitness after having a metal hip implanted in January in a bid to save his career.
He pulled out of the doubles at the US Open to concentrate on his singles career.