Ted Cruz was booed by furious delegates for refusing to endorse Donald Trump, undermining calls for Republican unity.
The remarkable scenes at the Republican National Convention overshadowed the speech by Indiana governor Mike Pence, who was formally nominated for vice-president.
Cruz, a Texas senator who was defeated by Trump in the race to become Republican candidate for the White House, spoke on the third day of the party's convention in Cleveland, Ohio.
He mentioned Trump by name only once, and stubbornly refused to endorse him, triggering thunderous boos from angry delegates as Cruz encouraged Americans to simply "vote your conscience" in November.
Trump - who will address the fourth and final day of the conference - unexpectedly walked into the arena just as Cruz was ending his remarks.
Delegates chanted Trump's name and implored Cruz to voice his support for the businessman, to no avail.
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 Trump's controversial campaign, and were at odds with the later claim by Pence that it was a "united party".
Cruz said: "Vote for candidates up and down the ticket who you trust to defend our freedom and to be faithful to the Constitution."
Trump and Cruz have had a complicated relationship throughout the presidential campaign. Cruz's speech at the convention drew a mixed response online.
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 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 Cruz's decision was "totally selfish".
Trump responded to the speech with a tweet.
Pence, an experienced politician and favourite of conservatives, lauded Trump as his own man, an independent spirit, and said change in the country will be "huge" under his presidency.
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 to 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.