Juventus fell to a surprise 3-2 defeat at Sampdoria, who maintained their perfect Serie A home record on Sunday.
Duvan Zapata, Lucas Torreira and Gian Marco Ferrari put Sampdoria 3-0 up in the second half before Juventus threatened an astonishing stoppage-time comeback with a penalty from Gonzalo Higuain and a strike from Paulo Dybala.
Juve, who take on Barcelona in the Champions League next week, are now four points behind leaders Napoli, who beat AC Milan 2-1 on Saturday.
Sampdoria, meanwhile, moved five points behind Juventus with a game in hand on most other teams.
The champions were without defender Andrea Barzagli and goalkeeper Gianluigi Buffon, who were recovering from the disappointment of Italy's failure to qualify for the World Cup, and Sampdoria made the most of their absences.
They broke the deadlock six minutes into the second half when a poor clearance by Federico Bernardeschi fell straight to Zapata, who sent a looping header past Wojciech Szczesny.
Juventus upped the pressure, but Sampdoria doubled their lead in the 71st thanks a fine strike by Torreira from just outside the area.
It got worse for Juve eight minutes later, when a cutback from Fabio Quagliarella was not dealt with and Ferrari tapped in from close range.
Juventus had penalty appeals turned down at the end of the first half but were awarded a spot kick at the end of the second when Douglas Costa was tripped by Ivan Strinic.
Higuain converted in the first minute of stoppage time and Dybala reduced the deficit further moments later when he ran down the right flank before cutting inside and placing the ball into the bottom corner.
But Juve did not have time to get the equaliser, with Higuain flagged offside in the last piece of action.