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The US Justice Department filed a lawsuit against California on Wednesday for allowing transgender athletes to compete in girls sports.
Female student athletes in California are being subjected to "unfair competition and reckless endangerment by male participation on female high-school sports teams," the department said.
The lawsuit accuses California of violating Title IX, the law that prohibits sex discrimination in educational programs that receive federal funding.
The Justice Department suit is the latest salvo in a showdown between the administration of Republican President Donald Trump and the Democratic-ruled state.
Trump sent thousands of National Guard troops to Los Angeles last month to quell protests against roundups of undocumented migrants by federal agents.
California Governor Gavin Newsom has said the troops were not necessary to address the mostly peaceful protests, but his legal efforts to have them removed have failed so far.
Trump threatened last month to impose "large scale" fines against California after a transgender high school athlete's victory at the state track and field championships.
The Justice Department suit accuses the California Department of Education and California Interscholastic Federation of engaging in "illegal sex discrimination against female student athletes by allowing males to compete against them."
"The Governor of California has previously admitted that it is 'deeply unfair' to force women and girls to compete with men and boys in competitive sports," Attorney General Pam Bondi said. "But not only is it 'deeply unfair,' it is also illegal under federal law."
The Justice Department sued Maine in April for allowing transgender athletes to compete in girls sports, and the Trump administration has moved to cut the northeastern state's federal funding for public schools.
The Supreme Court agreed earlier this month to hear cases next term challenging state laws in Idaho and West Virginia banning transgender athletes from female competitions.
More than two dozen US states have passed laws in recent years barring athletes who were assigned male at birth from taking part in girls or women's sports.
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