The lawsuit filed in a federal court in San Francisco argues that the policy is unconstitutional.
Women can serve in front line positions in the US military, but they are barred from ground combat units.
A Pentagon spokesman said 14,500 combat positions had been opened to women under the current defence secretary.
He added that Defence Secretary Leon Panetta had "directed the services to explore the possibility of opening additional roles for women in the military".
But ACLU lawyer Ariela Migdal said the changes so far were "not enough". The lawsuit argues that continuing restrictions violate servicewomen's constitutional rights to equality.
One of the plaintiffs, Marine Corps Capt Zoe Bedell, said existing rules had blocked her advancement in the Marines.
"The military is the last place where you are allowed to be discriminated against because of your gender," she said.
Women account for about 14% of the 1.4 million active US military personnel.
The lawsuit launched on Tuesday says they are barred from 238,000 positions, but also alleges that they are already serving unofficially in combat units.
The American Civil Liberties Union has filed a challenge on behalf of four US servicewomen against a ban on women being deployed in most combat roles.