A variety of molecules expressed at the midline of the developing central nervous system (CNS) control multiple aspects of pattern formation and axon guidance. We recently identified monoclonal antibody (mAb) CARO 2 as a novel marker of the ventral midline in the developing rat CNS, and the corresponding antigen as a membrane-associated 28-kDa protein. We report here the isolation of cDNA clones encoding the mAb CARO 2 antigen, which we rename VEMA, for ventral midline antigen. The deduced amino acid sequence of VEMA contains a single transmembrane domain near its N-terminus and several tyrosine-based internalization motifs. These structural features are consistent with the association of VEMA to intracellular membranes. In situ hybridization analyses demonstrate that VEMA mRNA is predominantly expressed at the ventral midline. The restricted distribution of VEMA, as well as several characteristics of its primary structure, suggest a role for this protein in regulating axon guidance in the mammalian CNS.