Protein patterns of mitochondrial outer membrane, inner membrane, and matrix from non-synaptic (free) mitochondria from rat cerebellum at different ages (4, 8, 12, 16, 20, and 24 months) were analyzed by gel electrophoresis. Acute L-acetylcarnitine treatment was performed by a single i.p. injection (100 mg/kg body weight) of the substance 60 min before the sacrifice of the animals. Different age-dependent changes were obtained for the proteins of the three fractions. The amount of some protein subunits increased and/or decreased after drug treatment. In particular, protein composition of the inner mitochondrial membrane showed significant age-related modifications. This result probably indicates differences in protein synthesis and/or turnover rates in the various mitochondrial compartments during aging. Acute L-acetylcarnitine treatment caused: a high increase in the amount of one inner membrane protein with Mw 16 kDa, at all the ages studied; a decrease in the amount of many other inner membrane proteins; modifications of some matrix proteins. Our results show that in vivo administration of L-acetylcarnitine affects mainly the inner membrane protein composition of cerebellar mitochondria.