The lawsuit, filed in federal court in San Francisco, is the second one this year over the 1994 rule that bars women from being assigned to ground combat units, which are smaller and considered more dangerous since they are often in battle for longer periods.
The legal effort comes less than a year after the ban on gays serving openly was lifted and as officials are surveying Marines about whether women would be a distraction in ground combat units.
"I'm trying to get rid of the ban with a sharp poke," said U.S. Army Staff Sgt. Jennifer Hunt, who was among the plaintiffs in the latest lawsuit and was injured in 2007 when her Humvee ran over an improvised explosive device in Iraq.
Hunt and the other three women said the policy unfairly blocks them from promotions and other advancements open to men in combat. Three of the women are in the reserves. A fourth, Marine Corp Lt. Colleen Farrell, leaves active duty this week.
Women comprise 14 percent of the 1.4 million active military personnel. The lawsuit alleges that women are barred from 238,000 positions across the Armed Forces.
At a Washington, D.C., news conference, Pentagon press secretary George Little said the Defense Department was making strides in allowing more women to experience combat. He said Defense Secretary Leon Panetta has opened about 14,500 combat positions to women.
"And he has directed the services to explore the possibility of opening additional roles for women in the military," Little said. "His record is very strong on this issue."
American Civil Liberties Union Ariela Migdal, who represents the four women, said Panetta's actions weren't enough. She called for an end to the combat ban. "These tweaks and minor changes on the margins do a disservice to all the women who serve," she said.
"It falls short," she said. "It is not enough."
Marine Corps Capt. Zoe Bedell said she left active duty, in large part, because of the combat exclusion policy. Bedell said she was frustrated that her advancement in the Marines was blocked by her inability to serve directly in combat units.
"The military is the last place where you are allowed to be discriminated against because of you gender," she said.
Bedell said the blurred front lines of modern warfare, with suicide bombs and sniper attacks, have put more and more women in combat situations.
More than 144 female troops have been killed and more than 860 have been wounded in Iraq and Afghanistan since the wars began, according to Pentagon statistics. Roughly 20,000 of the 205,000 service members currently serving in Afghanistan are women.
Military leaders say they want to make sure lifting gender-based barriers would not disrupt the cohesion of the smaller combat ground units and military operations.
The Marine Corps' top leader, Gen. James Amos, ordered a survey of 53,000 troops to get their views, including whether they believe women in those units would distract male Marines from doing their jobs. The results have not been released yet.
The lawsuit alleges the ban violates constitutional female service members' equal rights. "As a direct result of this policy," the lawsuit states, "women - as a class and solely because of their gender - are barred from entire career fields.
The lawsuit also alleges that women are already serving unofficially in combat units.
Air National Guard Major Mary Jennings Hegar sustained shrapnel wounds in 2009 when she exchanged fire on the ground in Afghanistan after her Medevac helicopter was shot down. Both she and Hunt received Purple Heart medals for their injuries.
The lawsuit was assigned to U.S. District Judge Edward Chen, an appointee of President Barack Obama.