PETA accuses SeaWorld of keeping five performing killer whales in conditions that violate the 13th Amendment ban on slavery.
The plaintiffs are the five orcas, Tilikum and Katina based at SeaWorld in Orlando, Fla., and Corky, Kasatka and Ulises at SeaWorld San Diego.
The lawsuit asks the court to order the orcas released to the custody of a legal guardian who would find a "suitable habitat" for them.
Former dolphin trainer Ric O'Barry said the law needs to change.
"The time is now to end slavery of orcas denied everything that's natural to them, exploited as virtual breathing machines and forced to perform for SeaWorld's profit," said O'Barry.
In a statement, SeaWorld called the accusations baseless.
"SeaWorld is among the world's most respected zoological institutions," the company said in a statement. "There is no higher priority than the welfare of the animals entrusted to our care and no facility sets higher standards in husbandry, veterinary care and enrichment."
The legal action is likely to stoke an ongoing, intense debate at America's law schools over expansion of animal rights.
The chances of the suit succeeding are slim, according to legal experts not involved in the case; any judge who hews to the original intent of the authors of the amendment is unlikely to find that they wanted to protect animals.
But PETA relishes engaging in the court of public opinion, as evidenced by its provocative anti-fur and pro-vegan campaigns.
Tilikum, a six-ton male, made national news in February 2010 when he grabbed a trainer at the close of a performance and dragged her underwater until she drowned.
The Associated Press contributed to this report.