Federal highway officials have rejected a D.C. application for $29 million to move a congressional plant nursery and build a parking facility over the planned Anacostia Metro subway station, raising further doubts about the future of the long-delayed "inner-city" Green Line.

City Transportation Department Director Tom Downs said the Federal Highway Administration informed him this week that it considered the Anacostia project to be rail-oriented and therefore not eligible for highway grants. The city plans to appeal, Downs said.

The denial could delay by two more years, to 1990, the planned opening of Green Line track between L'Enfant Plaza and Anacostia, Downs said. Rising construction costs and declining federal funding programs have fueled speculation that large segments of the largely unstarted Green Line never will be built.

The grant is crucial to Green Line construction south of L'Enfant Plaza, Downs said, because it includes funds to relocate the station site's current occupant, a nursery operated by the Architect of the Capitol to provide plants and flowers for the Hill.

If the site is not vacated, work on the station and tunneling beneath the Anacostia River, scheduled to begin next year, would be delayed, Downs said. Not getting the grant would "just wreck the next two years," he said.

Metro planners already had tentatively postponed the segment's target opening date from 1986 to 1988, because of expected shortfalls in federal funds. Officials also have expressed concern over the building schedule because there still is no firm agreement with the architect's office over where the nursery will be relocated, even if the money becomes available.

The Anacostia parking facility's design shows spaces for 1,300 cars and platforms where 1,000 busloads of commuters would move on and off rail cars daily. The District argues that it is a "rubber tire" project and should get highway funds. Much of the cost is for access roads and ramps.

With the Reagan administration trimming capital programs of the Urban Mass Transportation Administration, Metro had hoped to stretch its share by tapping interstate highway funds to pay for the parking facility.

If D.C.'s appeals to the Federal Highway Administration fail, Downs said, it may be necessary to renegotiate the four-year construction program that Metro's eight member jurisdictions signed in October following months of often bitter discussion. That would be necessary to finance the parking project with mass transit grants.

Yesterday, Downs informed area transit officials of the federal decision at Metro's annual policy conference at the Airlie retreat center near Warrenton and asked them to actively support the appeal. Lining up help from members of the area's congressional delegation also was discussed.

D.C. hopes the Green Line will revitalize declining neighborhoods of the inner city. So far, only one station south of L'Enfant Plaza, Waterfront, has been built, with nothing completed north of Gallery Place.

The threat to the Anacostia station is the latest setback to D.C. efforts to speed construction of the Green Line. In talks leading up to the October construction agreement this year, city representatives pushed hard for a bigger share of Metro's 1982 capital budget. In those talks, the local governments assumed they would get $315 million in federal capital funds but now expect $290 million or less.

As a result, D.C. officials now concede they will have to give up a $15 million contract contained in the October agreement to begin the first tunneling around the U Street station next year.