Ciro Immobile scored twice in a 2-1 Lazio victory in Turin as Paulo Dybala failed to save Juventus from suffering their first home defeat in Serie A in more than two years.
The defeat meant that titleholders Juventus dropped five points behind leaders Napoli who won their eighth straight game by beating bitter rivals AS Roma 1-0 away with a Lorenzo Insigne goal, although last season's runners-up hit the woodwork twice.
Ciro Immobile scored twice early in the second half for Lazio, the second goal a penalty, after Douglas Costa had given the champions a halftime lead.
Juve's last home defeat was against Udinese in August, 2015 and they have since been unbeaten at their own stadium for 57 matches, including 41 in Serie A.
There was extraordinary drama in stoppage time when Dybala hit the foot of the post and before Juve were awarded a penalty.
Lazio substitute Gil Patric clattered into Federico Bernardeschi and, although he touched the ball, he also raised his leg and upended the Juventus player.
The tackle led to scuffles between players while referee Paolo Silvio Mazzoleni consulted the video assistant before pointing to the spot amid furious Lazio protests.
However, Dybala's effort with the last kick of the match was brilliantly saved by Thomas Strakosha.
Juve's first league defeat of the season left them on 19 points from eight games, level with Inter and Lazio, while Napoli have 24.
Costa put Juventus ahead in the 23rd minute from a rebound after Strakosha parried Sami Khedira's header, the goal being confirmed after the referee consulted the VAR.
Juventus nearly got another in an odd incident when Strakosha's clearance off his line hit Gonzalo Higuain and rebounded on to the crossbar from point-blank range.
Immobile levelled two minutes into the second half after a one-two with Luis Alberto, then won and converted a penalty seven minutes later, after he was upended by Buffon, for his eleventh league goal of the season.
"We have to avoid these lapses in concentration," said Juventus coach Massimiliano Allegri whose side are aiming for a seventh successive title. "To win the championship, we have to fight all the way."
Insigne scored Napoli's winner after 20 minutes with a cool finish after the ball rebounded to him off a Roma player.
Roma pressed desperately for an equaliser in the final 20 minutes and Federico Fazio had a header tipped on to the post by Napoli goalkeeper Pepe Reina before Edin Dzeko saw another header bounce off the top of the crossbar.
[attach id=606538 size="large" align="left"]Lazio goalkeeper Thomas Strakosha is mobbed his team-mates after saving Paulo Dybala's penalty.[/attach]