Hemocyanin-conjugated peptides corresponding to amino acid sequences from 14 different regions of the rat dopamine transporter were used to immunize rabbits and the resulting antisera were tested for dopamine transporter recognition. Eleven of the peptide sequences produced antipeptide antibodies detectable with ELISA assays. Sera directed against four of the peptides immunoprecipitated striatal transporters photoaffinity labeled with [(125)I]DEEP. Antisera directed against a peptide sequence at the C-terminal region of the transporter's putative second extracellular loop, "peptide 5," recognized photolabeled dopamine transporter protein from rat, dog, and human basal ganglia. These sera also recognized dopamine transporters from rat striatum in immunoblots; however, no reaction was found when rat cerebellum or frontal cortical tissue was used. These results provide further evidence that the cloned cDNA and its deduced amino acid sequence represents the protein expressed in brain and highlights the regions of the transporter which may be promising sites for the development of high-affinity antibodies.