Gatlin clocked 9.94 seconds, and Bolt crossed second in 9.95 in Rome.
It marked Bolt's first significant loss since he was disqualified for a false start at the 2011 world championships in Daegu, South Korea. Bolt was also beaten by Tyson Gay in Stockholm in 2010.
Gatlin has won all five of his 100 races this year. He's shaping up as a serious threat to Bolt at worlds in August in Moscow.
Bolt, the six-time Olympic champion from Jamaica, also struggled in his only other 100 this year. He narrowly won in a relatively slow 10.09 in the Cayman Islands last month.
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