Mammalian NADH:ubiquinone oxidoreductase (complex I) is made up of at least 46 subunits and is one of the largest enzyme complexes known. It catalyzes the first step of the respiratory electron transport chain through the oxidation of NADH, providing two electrons for the reduction of ubiquinone to ubiquinol, thus propelling protons across the inner membrane of the mitochondria, which subsequently drive ATP synthesis. Dysfunction of complex I has been implicated in various neurodegenerative disorders, and it is probably the most vulnerable component of the electron transport chain to inhibition by reactive oxygen species. We describe a simple spectrophotometric method for estimating the activity of complex I from mitochondria isolated from regions of the central nervous system of mice.