L-carnitine stimulated CO2 production from 1-14C pyruvate in mitochondria from human skeletal muscle nearly twofold. A comparable increase in the pyruvate dehydrogenase complex (PDHC) activity was seen. Moreover, in the presence of L-carnitine and at pyruvate concentration greater than 0.25 mM, this effect was associated with a marked increase of acetylcarnitine synthesis. Deoxycarnitine, an inhibitor of carnitine acetyltransferase (CAT), partially reversed the effect of carnitine on PDHC activity. The stimulatory effect of carnitine on PDHC activity in human mitochondria is mediated by the modulation of the intramitochondrial acetyl-CoA/CoASH ratio.