We explored the protective mechanisms of human neuronal mitochondrial uncoupling protein-5 (UCP5) in MPP+- and dopamine-induced toxicity after its stable overexpression in SH-SY5Y cells. We raised specific polyclonal antibodies. Overexpressed UCP5 localized in mitochondria but not in cytosol. UCP5 overexpression increased proton leak, decreased mitochondrial membrane potential (MMP), reduced ATP production, and increased overall oxygen consumption (demonstrating uncoupling activity). UCP5 overexpression did not affect other neuronal UCP expression (UCP2 and UCP4). Overexpressing UCP5 is protective against MPP+- and dopamine-induced toxicity. MPP+ and dopamine exposure for 6h reduced MMP and increased superoxide levels. ATP levels in UCP5-overexpressing cells were preserved under MPP+ and dopamine toxicity, comparable to levels in untreated vector controls. At 24h, UCP5 overexpression preserved MMP, ATP levels, and cell survival, attenuated superoxide generation, and maintained oxidative phosphorylation as indicated by lower lactate levels. MPP+ and dopamine exposure induced UCP5 mRNA transcription but did not decrease transcript degradation, as inhibition of transcription by actinomycin-D abolished induction by either toxin. Compared with our previous studies on UCP4, we observed functional differences between UCP4 and UCP5 in enhancing mitochondrial efficiency. These neuronal UCP homologues may work synergistically to maintain oxidative balance (through uncoupling activities) and ATP production (by modifying MMP). © 2010 Elsevier Inc.