Regulation of β-oxidation under various metabolic conditions and energy loads was studied by employing a newly developed method for assaying 3-hydroxyacyl-CoA and 2- trans-enoyl-CoA intermediates of fatty acid oxidation. A 66% inhibition of oleate oxidation with a concomitant 68% inhibition of oxygen consumption resulted in a 81% decrease in the carnitine/acyl-carnitine ratio, but the concentrations of 2- trans-enoyl-CoA and 3-hydroxyacyl-CoA esters did not change significantly and the acid-insoluble acyl-CoA content did not change. The acetyl-CoA concentration increased three-fold, however, and there was a simultaneous tendency for the NADH NAD + ratio to shift towards oxidation. The results suggest that the main regulatory site of fatty acid oxidation resides at an early step in the pathway. Since the concentrations of the acyl-CoA derivatives identified did not undergo major changes even though the acyl-carnitine concentration changed some rate limitation must already occur at the steps of acyl transport into the mitochondria or the carnitine acyltransferase II.