The dopamine transporter/cocaine receptor (DAT) is the site at which cocaine exerts rewarding/reinforcing effects. Recent cloning of the DAT cDNA allows structure/function relationships of the DAT to be sought by mutating selected residues and examining influences on cocaine analog recognition and dopamine transport. Mutations preferentially affecting one of these processes provide novel direct evidence for the possibility that anticocaine medications could differentially impact one of these processes but not the other. Because of work defining the amino acids important for catecholamine recognition by catecholamine receptors, and data suggesting that polar regions of cocaine are important for transporter recognition of this molecule, polar residues in transmembrane segments were mutated. Knowledge about cysteine residues would help to understand the molecule's structure. Substitution of aspartate 79 dramatically reduced uptake of dopamine and MPP+ and the mutants' affinity for CFT without affecting B(max). Replacement of the serines in the 7th hydrophobic region reduced dopamine and MPP+ uptake, while sparing CFT binding. Changing serines in the 8th hydrophobic region has no effects. These results demonstrate that aspartate and serine residues lying within putative transmembrane regions are crucial for DAT function, and provide the first identification of residues differentially important for cocaine binding and for dopamine uptake. Elucidating the structures of the putative extracellular domains is also important. Substitution for the cysteines in the DAT second hydrophobic putative extracellular domain selectively and interestingly decreases the function of mutant transporters, conversely, substitution for either of two other cystines had no significant influence. Features of transporter secondary structure are thus important for transporter function.