Synthesis of [3H]acetylcholine from [3H]acetyl-L-carnitine was demonstrated in vitro by coupling the enzyme systems choline acetyltransferase and carnitine acetyltransferase. Likewise, both [3H] and [14C] labeled acetylcholine were produced when [3H]acetyl-L-carnitine and D-[U-14C] glucose were incubated with synaptosomal membrane preparations from rat brain. Transfer of the acetyl moiety from acetyl-L-carnitine to acetylcholine was dependent on concentration of acetyl-L-carnitine and required the presence of coenzyme A, which is normally produced as an inhibitory product of choline acetyltransferase. These results provide further evidence for a role of mitochondrial carnitine acetyltransferase in facilitating transfer of acetyl groups across mitochondrial membranes, thus regulating the availability in the cytoplasm of acetyl-CoA, a substrate of choline acetyltransferase. They are also consistent with a possible utility of acetyl-L-carnitine in the treatment of age-related cholinergic deficits.