Cocaine blocks the normal role of the dopamine transporter (DAT) in terminating dopamine signaling through molecular interactions that are only partially understood. Cocaine analog structure-activity studies have suggested roles for both cationic and aromatic interactions among DAT, dopamine, and cocaine. We hypothesized that phenylalanine residues lying in putative DAT transmembrane (TM) domains were good candidates to contribute to aromatic and/or cationic interactions among DAT, dopamine, and cocaine. To test this idea, we characterized the influences of alanine substitution for each of 29 phenylalanine residues lying in or near a putative DAT TM domain. Cells express 22 mutants at near wild-type levels, manifest by DAT immunohistochemistry and binding of the radiolabeled cocaine analog [(3)H](-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (CFT). Seven mutants fail to express at normal levels. Four mutations selectively reduce cocaine analog affinities. Alanine substitutions at Phe(76), Phe(98), Phe(390), and Phe(361) located in TM domains 1 and 2, the fourth extracellular loop near TM 4 and in TM 7, displayed normal affinities for dopamine but 3- to 8-fold reductions in affinities for CFT. One TM 3 mutation, F(155)A, selectively decreased dopamine affinity to less than 3% of wild-type levels while reducing CFT affinity less than 3-fold. In a current DAT structural model, each of the residues at which alanine substitution selectively reduces cocaine analog or dopamine affinities faces a central transporter cavity, whereas mutations that influence expression levels are more likely to lie at potential helix/helix interfaces. Specific, overlapping sets of phenylalanine residues contribute selectively to DAT recognition of dopamine and cocaine.