Abnormalities of mitochondrial energy metabolism may play a role in normal aging and certain neurodegenerative disorders. In this regard, complex I of the electron transport chain has received substantial attention, especially in Parkinson's disease. The conventional method for studying complex I has been quantitation of enzyme activity in homogenized tissue samples. To enhance the anatomic precision with which complex I can be examined, we developed an autoradiographic assay for the rotenone site of this enzyme. [3H]dihydrorotenone ([3H]DHR) binding is saturable (KD = 15-55 nM) and specific, and Hill slopes of 1 suggest a single population of binding sites. Nicotinamide adenine dinucleotide (NADH) enhances binding 4- to 80-fold in different brain regions (EC50 = 20-40 microM) by increasing the density of recognition sites (Bmax). Nicotinamide adenine dinucleotide phosphate also increases binding, but NAD+ does not. In skeletal muscle, heart, and kidney, binding was less affected by NADH. [3H]DHR binding is inhibited by rotenone (IC50 = 8-20 nM), meperidine (IC50 = 34-57 microM), amobarbitol (IC50 = 375-425 microM), and MPP+ (IC50 = 4-5 mM), consistent with the potencies of these compounds in inhibiting complex I activity. Binding is heterogeneously distributed in brain with the density in gray matter structures varying more than 10-fold. Lesion studies suggest that a substantial portion of binding is associated with nerve terminals. [3H]DHR autoradiography is the first quantitative method to examine complex I with a high degree of anatomic precision. This technique may help to clarify the potential role of complex I dysfunction in normal aging and disease.