Several analogs of 1-methyl-4-phenylpyridinium (MPP+) were evaluated for their affinity for the dopamine uptake system and their ability to inhibit NADH dehydrogenase (complex I) of the mitochondrial electron-transport chain. Moreover, these compounds were tested for their ability to cause selective dopaminergic neurotoxicity in cultured mesencephalic neurons. Simultaneous [3H]dopamine and gamma-amino-[14C]butyric acid uptake and immunocytochemical techniques were used as indices of neuronal damage in cultured cells. The compounds that were potent and selective dopaminergic neurotoxins had high affinity for the dopamine transport system, as measured by their ability to cause dopamine release, and were similar to MPP+ in inhibiting mitochondrial respiration. One compound (1-methyl-4-phenylpyrimidinium) had high affinity for the dopamine uptake system but was a weak inhibitor of mitochondrial respiration and, accordingly, was not neurotoxic. The 4'-alkylated analogs of MPP+, which were poor substrates for the dopamine uptake system and extremely potent inhibitors of mitochondrial respiration, caused a nonselective damage of neurons in culture. Analogs that were not substrates for the dopamine carrier and not inhibitors of mitochondrial respiration were not neurotoxic. This study describes the neurotoxicity of a number of analogs of MPP+ and highlights the importance of the dopamine uptake system and the ability to inhibit mitochondrial respiration as critical processes in conferring selectivity and neurotoxicity, respectively, to MPP+ and analogs, for dopaminergic neurons in culture.