Analyses of mutation effects can aid in understanding how large proteins act. The dopamine transporter (DAT) mediates complex actions in recognizing cocaine and in recognizing and translocating dopamine, sodium, and chloride. DAT proline residues, especially those in transmembrane (TM) domains, are good candidates for involvement in these DAT actions. We now report production of mutants substituting alanine and/or glycine residues for 16 prolines located in or near putative DAT TM domains. We examine effects of these modifications on DAT expression, dopamine uptake, and cocaine analog binding. Mutants in prolines located in five DAT TM domains and four connecting loops alter apparent DAT membrane targeting. Five mutations decrease dopamine affinities more than threefold without significantly decreasing cocaine analog affinities. One decreases cocaine analog affinity without decreasing dopamine affinity. Two mutations decrease affinities for both dopamine and cocaine analog. P101 is especially implicated in dopamine uptake. Alanine substitution for this proline yields dopamine V(max) values of less than 3% of wild-type values despite dopamine affinities more than fourfold higher than wild-type and normal Na(+) and Cl(-) dependence. These DAT proline mutants identify DAT regions likely for dopamine translocation and for recognition of dopamine and cocaine.