Defects in mitochondrial energy metabolism have been implicated in several neurodegenerative disorders. Defective complex I (NADH:ubiquinone oxidoreductase) activity plays a key role in Leber's hereditary optic neuropathy and, possibly, Parkinson's disease, but there is no way to assess this enzyme in the living brain. We previously described an in vitro quantitative autoradiographic assay using [(3)H]dihydrorotenone ([(3)H]DHR) binding to complex I. We have now developed an in vivo autoradiographic assay for complex I using [(3)H]DHR binding after intravenous administration. In vivo [(3)H]DHR binding was regionally heterogeneous, and brain uptake was rapid. Binding was enriched in neurons compared with glia, and white matter had the lowest levels of binding. In vivo [(3)H]DHR binding was markedly reduced by local and systemic infusion of rotenone and was enhanced by local NADH administration. There was an excellent correlation between regional levels of in vivo [(3)H]DHR binding and the in vitro activities of complex II (succinate dehydrogenase) and complex IV (cytochrome oxidase), suggesting that the stoichiometry of these components of the electron transport chain is relatively constant across brain regions. The ability to assay complex I in vivo should provide a valuable tool to investigate the status of this mitochondrial enzyme in the living brain and suggests potential imaging techniques for complex I in humans.