The dopamine (DA) and norephinephrine (NE) transporters demonstrate important differences in their selectivity for catecholamines and the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+), yet their primary sequences and predicted topology are strikingly similar. To delineate discrete structural domains contributing to pharmacologic and kinetic differences between the DA and NE transporters, a series of recombinant chimeras was generated by a restriction site-independent method and expressed in mammalian cells. Functional analyses of the chimeras delineate two discrete regions spanning the first through the third transmembrane domains (TM1-3) and TM10-11 that contribute to differences in their apparent affinities for DA, NE, and MPP+. These studies also suggest that TM2-3 of the DA transporter have a role in selectively increasing the rate of DA uptake as compared with NE. TM4-8 of the DA transporter may influence the relative rate with which MPP+ is taken up into cells and could contribute to its selective toxicity in neurons expressing the DA transporter. These structure-function studies using chimeras of members of the superfamily of Na(+)- and Cl(-)-dependent transporters provide a framework for identifying the specific structural or regulatory determinants contributing to substrate recognition and translocation by the DA and NE transporters.