The regulation of fatty acid oxidation in isolated myocytes was examined by manipulating mitochondrial acetyl-CoA levels produced by carbohydrate and fatty acid oxidation. L-carnitine had no effect on the oxidation of [U-14C]glucose, but stimulated oxidation of [1-14C]palmitate in a concentration-dependent manner. L-carnitine (5 mM) increased palmitate oxidation by 37%. The phosphodiesterase inhibitor, enoximone (250 microM), also increased palmitate oxidation by 51%. Addition of L-carnitine to enoximone resulted in a two-fold increase of palmitate oxidation. Whereas, dichloroacetate (DCA, 1 mM), which stimulates PDH activity, decreased palmitate oxidation by 25%. Furthermore, the addition of DCA to myocytes preincubated with either L-carnitine or enoximone, had no effect on the carnitine-induced stimulation of palmitate, and reduced that of enoximone by 50%. Varied concentrations of DCA decreased the oxidation of palmitate and octanoate; but increased glucose oxidation in myocytes. The rate of efflux of acetylcarnitine was highest when pyruvate was present in the medium compared to efflux rates in presence of palmitate or palmitate plus glucose. Although the addition of L-carnitine plus enoximone resulted in a two-fold increase in palmitate oxidation, acetylcarnitine efflux was minimal under these conditions. Acetylcarnitine efflux was highest when pyruvate was present in the medium. These rates were dramatically decreased when myocytes were preincubated with enoximone, despite the stimulation of palmitate oxidation by this compound. These data suggest that: (1) fatty acid oxidation is influenced by acetyl-CoA produced from pyruvate metabolism; (2) L-carnitine may be specific for mitochondrial acetyl-CoA derived from pyruvate oxidation; and (3) it is probable that acetyl-CoA from beta-oxidation of fatty acids is directly channeled into the citric acid cycle.