The star, whose "Born This Way" album recently topped one million sales in a week, delighted tens of thousands of people at a brief concert in the vast field where the ancient Roman masses would gather for spectacles.
Wearing a green wig, she played the piano and sang a few numbers. But she devoted much of her appearance after an annual European gay pride parade to denounce intolerance and discrimination against gays and transgender people. Among the places she cited was the Middle East, Poland, Russia and Lithuania.
Lady Gaga told the crowd she is often asked "How gay are you, Lady Gaga?"
"My answer is: 'I am a child of diversity."'
She also proudly cited her Italian roots - saying she was really named Stefania Giovanna Angelina Germanotta.
And she told fans her costume - a sleek black top with one bare shoulder and billowing plaid skirt - were from the last collection of Gianni Versace.
Decrying intolerance of homosexuality, Lady Gaga lamented that young people who are gay are susceptible to "suicide, self-loathing, isolation."
Many in the crowd had participated in an hours-long parade of colourful floats and brightly costumed marchers through Rome's historic centre before the rally. The events were part of the annual Europride day to encourage gay rights on the continent.
Lady Gaga praised her audience for its "great courage" which she says inspires her.
Europride organisers hope the event will draw attention to discrimination gays face in many parts of the world. The US ambassador was among those who invited Lady Gaga to Rome.
"I am so honoured to be here," Lady Gaga said, recalling how, earlier in the day, she lay naked in silk sheets in her hotel room and enjoyed the din of adoring fans and packs of photographers in the street below.
Organisers said Rome was a significant choice of venue, since it is home to the Vatican, which staunchly opposes legislation that would recognise same-sex marriage or adoption by gay couples.
Others hoped the turnout would send a message to Premier Silvio Berlusconi, the Italian leader dogged by a sex scandal involving an alleged 17-year-old Moroccan prostitute. The billionaire media mogul triggered outrage from gay rights groups last fall when he contended during a public appearance that it was "better to be passionate about a beautiful girl than a gay."
Berlusconi's equal opportunity minister, a woman, defended the premier, saying he had just been joking and had no intention of offending gays. A government undersecretary further provoked protests when she said she was sure "all Italian parents hope to have heterosexual children."
The premier, who is on trial in Milan for allegedly paying the teenager for sex and then using his office to try to cover it up, has denied any wrongdoing.
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