Ted Cruz was booed by furious delegates for refusing to endorse Donald Trump, undermining calls for Republican unity.
The remarkable scenes overshadowed the speech by Indiana Governor Mike Pence, who was formally nominated for vice-president.
Mr Cruz, a Texas senator, was defeated by Mr Trump in the race to become Republican candidate in the race for the White House, and spoke on the third day of the party's convention in Cleveland, Ohio.
He mentioned Mr Trump by name only once, and stubbornly refused to endorse him, triggering thunderous boos from angry delegates as he encouraged Americans to simply "vote your conscience" in November.
Mr Trump - who will address the fourth and final day of the conference - unexpectedly walked into the arena just as Mr Cruz was ending his remarks.
Delegates chanted Mr Trump's name and implored Mr Cruz to voice his support for the businessman, to no avail.
Mr Cruz's wife Heidi had to be escorted from the convention floor for her own safety as she was heckled by Trump delegates.
The extraordinary scenes show how split Republicans are following Mr Trump's controversial campaign, and were at odds with the later claim by Mr Pence that it was a "united party".
Mr Cruz said: "Vote for candidates up and down the ticket who you trust to defend our freedom and to be faithful to the Constitution."
Mr Trump and Mr Cruz have had a complicated relationship throughout the presidential campaign.
Mr Cruz first allied himself with the tycoon, banking that Mr Trump's campaign would collapse and he would be able to scoop up his supporters.
But as Mr Trump rattled off primary wins, tensions rose between the candidates.
Mr Trump sparked rumours about Mr Cruz's wife and father. Mr Cruz criticised Mr Trump as a "pathological liar" and "utterly amoral". Mr Trump began calling Mr Cruz "Lyin' Ted" at every opportunity.
Mr Cruz arrived in Cleveland with an eye on his own political future, holding a rally with hundreds of supporters who greeted him with chants of "2020" - suggesting his backers have no interest in seeing Mr Trump become a two-term president.
His defiance ripped open party divisions anew, on the summer's biggest political stage.
Trump allies were infuriated, including New Jersey Governor Chris Christie, who said Mr Cruz's decision was "totally selfish".
However in a late night tweet, Mr Trump said he had seen Mr Cruz's speech two hours before he took the stage but let him speak anyway, adding: "No big deal!"
Wow, Ted Cruz got booed off the stage, didn't honor the pledge! I saw his speech two hours early but let him speak anyway. No big deal!— Donald J. Trump (@realDonaldTrump) July 21, 2016
Mr Pence, an experienced politician and favourite of conservatives, lauded Mr Trump as his own man, an independent spirit, and said change in the country will be "huge" under his presidency.
He called presumptive Democratic nominee Hillary Clinton the "secretary of the status quo," referring to her former job as Secretary of State.
And he said he never thought he would be standing on the stage at his party's national convention, joking that Mr Trump is charismatic and must have been looking for balance in choosing him.
Mr Trump's family also kept up their efforts to rebrand the brash candidate as a warm husband and father.
Eric Trump, the candidate's 32-year-old son, took the stage near the evening's end, praising his father as other family members had earlier in the week.
"Vote for the one candidate who does not need this job," he said.