The celebrity pair were pictured looking irate during a break in auditions at The Lowry Theatre in Salford on Thursday.
Holden, 47, appeared to be shouting at McPartlin as fellow judge Alesha Dixon stood awkwardly alongside them.
They were interrupted by a member of the Britain’s Got Talent production team who placed her hand on Holden’s arm before they took part in the photocall.
But a source close to Holden claimed the pair were simply “chatting”.
Speaking to Mail Online they said: “This picture was taken whilst they were posing in front of hundreds of fans and press.
“There simply wasn’t any form of heated discussion whatsoever.”
A representative for McPartlin also shot down talk of an argument, branding it “absolute rubbish”.
McPartlin recently made his return to work following his split from wife Lisa Armstrong.
The Saturday Night Takeaway host told fans he was “very sad” to announce the news they are filing for a divorce after 11 years of marriage.
In a statement his spokesperson told the Standard: “In response to the recent speculation in the media, Ant is very sad to announce that, after eleven years, he is ending his marriage to Lisa.
“Ant asks for privacy at this difficult time, for both himself, Lisa and their immediate families.”
McPartlin is reportedly set to pay Armstrong a record £31 million in their divorce settlement to avoid a lengthy court battle.
The estranged pair are also expected to fight for custody over their five-year-old dog Hurley who was granted special dispensation to visit McPartlin in rehab last year.
Britain’s Got Talent returns to ITV later this year