In an attempt to elucidate the mechanism by which the rate of fatty acid oxidation is tuned to the energy demand of the heart, the effects of changing intramitochondrial ratios of [acetyl-CoA] [CoASH] and [NADH] [NAD+] on the rate of β-oxidation were studied. When 10 mm l-carnitine was added to coupled rat heart mitochondria to lower the ratio of [acetyl-CoA] [CoASH], the rate of palmitoylcarnitine β-oxidation, as measured by the formation of acid-soluble products, was stimulated more than fourfold at state 4 respiration while β-oxidation at state 3 respiration was hardly affected. Neither oxaloacetate nor acetoacetate, added to mitochondria to lower the [NADH] [NAD+] ratio, stimulated β-oxidation. Rates of respiration at states 3 and 4 were unchanged by additions of l-carnitine, oxaloacetate, or acetoacetate. Determinations of intramitochondrial ratios of [acetyl-CoA] [CoASH] by high performance liquid chromatography yielded values close to 10 for palmitoylcarnitine-supported respiration at state 4 and 2.5 at state 3 respiration. Addition of 10 mm l-carnitine caused a dramatic decrease of these ratios to less than 0.2 at both respiration states. Studies with purified or partially purified enzymes revealed strong inhibitions of 3-ketoacyl-CoA thiolase by acetyl-CoA and of l-3-hydroxyacyl-CoA dehydrogenase by NADH. Moreover, the activity of 3-ketoacyl-CoA thiolase at concentrations of acetyl-CoA and CoASH prevailing at state 3 respiration was 4 times higher than its activity in the presence of acetyl-CoA and CoASH observed at state 4. Altogether, this study leads to the conclusion that the rate of β-oxidation in heart can be regulated by the intramitochondrial ratio of [acetyl-CoA] [CoASH] which reflects the energy demand of the tissue. The thiolytic cleavage catalyzed by 3-ketoacyl-CoA thiolase may be the site at which β-oxidation is controlled by the [acetyl-CoA] [CoASH] ratio. © 1991.