Madonna was joined on stage by Britney Spears during her show at the Dodgers Stadium in Los Angeles on Thursday.
Dressed in a white shirt and black wide-leg trousers, Spears walked on stage to rapturous applause during Madonna's song Human Nature.
She sang a few lines before Madonna spun her around and kissed her hand.
Later on in the show, Spears' former boyfriend Justin Timberlake joined Madonna on stage to sing 4 Minutes with the 50-year-old pop star.
Spears had been rumoured to appear at the MTV Europe Music Awards in Liverpool on the same night.
Although she did not attend, the star still scooped two gongs including act of the year and album of the year for Blackout.
The singer accepted her awards with recorded video messages.
Spears and Madonna's most famous appearance on stage together was at the MTV Video Music Awards in 2003.
The pair made when headlines when they kissed on stage.
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