At low micromolar concentrations, 1-methyl-4-phenylpyridinium (MPP+), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively kills nigrostriatal dopaminergic neurons by mechanisms believed to involve impairment of mitochondrial complex I. A human neuroblastoma cell line expressing the dopamine transporter (DAT) was utilized to examine the effects of MPP+ on acute physiologic responses and subsequent cell death. Acute responses were measured by microphysiometry and by monitoring mitochondrial membrane potential with [3H]tetraphenylphosphonium (TPP+) uptake. MPP+ (10 microM) increased extracellular proton excretion in DAT-expressing cells within 2-3 min, but had no effect in untransfected cells. The lipophilic complex I inhibitor, rotenone, increased proton excretion in both cell lines. In DAT-expressing cells, mitochondrial membrane potential was reduced within I h of 10 microM MPP+ exposure. Rotenone reduced mitochondrial membrane potential in both cell lines. MPP+ caused apoptotic death of DAT-transfected cells 2-3 days after drug application, but did not kill untransfected cells. Thus, MPP+ produces immediate mitochondrial impairment only in cells that express DAT, and these changes occur days before overt cellular toxicity. The magnitude, time course and nature of these changes were similar to those produced by rotenone, confirming the site of action of MPP+ as mitochondrial complex I. These immediate mitochondrial effects appear to be an accurate predictor of subsequent cell death.