Outer segments of mammalian rod photoreceptor cells contain an abundantly expressed membrane protein that migrates with an apparent molecular mass of 220 kDa by SDS-gel electrophoresis. We have purified the bovine protein by immunoaffinity chromatography, determined its primary structure by cDNA cloning and direct peptide sequence analysis, and mapped its distribution in photoreceptors by immunocytochemical and biochemical methods. The full-length cDNA encodes a 2280-amino acid protein (calculated molecular mass of 257 kDa) consisting of two structurally related, tandem arranged halves. Each half consists of a hydrophobic domain containing six putative transmembrane segments followed by an ATP-binding cassette. A data base homology search showed that the rod outer segment 220-kDa protein is 40-50% identical in amino acid sequence to the ABC1 and ABC2 proteins cloned from a mouse macrophage cell line. Photoaffinity labeling with 8-azido-ATP and nucleotide inhibition studies confirmed that both ATP and GTP bind to this protein with similar affinities. Concanavalin A labeling and endoglycosidase H digestion indicated that the rod outer segment protein contains at least one carbohydrate chain. Immunocytochemical and biochemical studies have revealed that the 220-kDa glycoprotein is distributed along the rim region and incisures of rod outer segment disc membranes. From these studies we conclude that the 220-kDa glycoprotein of bovine rod outer segment disc membranes or Rim ABC protein is a new member of the superfamily of ABC transporters and is the mammalian homolog of the frog photoreceptor rim protein.