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Madonna claims Lady Gaga snubbed the opportunity to perform with her.
The singer once again took aim at her rival during a concert as part of her MDNA world tour in Minnesota, US, on Sunday.
Madonna told her crowd: "I have the best fans in the whole world. So take that, Lady Gaga.
"You know, I invited her to sing with me on stage but she turned me down. It's okay. I've been rejected before. It's good to build character."
The comments come just a week after the singer complimented Gaga a US talk show, saying she had a "good voice".
Madonna, 54, has criticized Gaga, 26, in the past over similarities between her song, 'Express Yourself', and the chorus to Gaga's 2011 hit, 'Born This Way'.
She said: "I thought, this is a wonderful way to redo my song. I mean, I recogniZed the chord changes. I thought it was... interesting."
However, when asked about the similarities between the tracks, Gaga said: "What a completely ridiculous thing to even question me about. If you put the songs next to each other, side by side, the only similarities are the chord progression. "It's the same one that's been in disco music for the last 50 years. Just because I'm the first f***ing artist in 25 years to think of putting it on Top 40 radio, it doesn't mean I'm a plagiarist, it means that I'm f***ing smart. Sorry."
Madonna has further aggravated the situation by playing a mash up of 'Express Yourself' and 'Born This Way' on the MDNA tour.