Cytochrome oxidase (CO), a mitochondrial energy-generating enzyme, contains both mitochondrial- and nuclear-encoded subunits. In neurons, local levels of CO activity vary among different neuronal compartments, reflecting local demands for energy. The goals of the present study were to determine if compartmental distribution of CO subunit proteins from the two genomes was correlated with local CO activity, and if their expression was regulated proportionately in neurons. The subcellular distributions of mitochondrial-encoded CO III and nuclear-encoded CO Vb proteins were quantitatively analyzed in mouse cerebellar sections subjected to postembedding immunocytochemistry. Local levels of subunit proteins were also compared to local CO activity, as revealed by CO cytochemistry. In order to study the regulation of subunit protein expression, we assessed changes in immunoreactivity of the two CO subunits as well as changes in CO activity in mouse superior colliculus after 1 to 7 days of monocular enucleation. We found that immunoreaction product for both CO III and CO Vb existed almost exclusively in mitochondria, but their compartmental distributions were different. CO III was nonhomogeneously distributed among different neuronal compartments, where its local level was positively correlated with that of CO activity. In contrast, the subcellular distribution of CO Vb was relatively uniform and did not bear a direct relationship with that of CO activity. Moreover, the two subunit proteins were disproportionately regulated by neuronal activity. CO III and CO activity exhibited parallel decreases after the deprivation of afferent input, and their changes were earlier and to a greater degree than that of CO Vb proteins. Thus, the present findings indicate that the local expression and/or distribution of CO subunit proteins from the two genomes may involve different regulatory mechanisms in neurons. Our data also suggest that the activity-dependent regulation of mitochondrial-encoded CO subunits is likely to play a major role in controlling the local levels of CO content and its activity.