The kinetics of bovine liver enoyl-CoA hydratase (EC 4.2.1.17) or crotonase with 2-trans-hexadecenoyl-CoA as a substrate were studied because different rates were obtained with two assay methods based on measurements of substrate utilization and product formation, respectively. L-3-Hydroxyhexadecanoyl-CoA, the product of the crotonase-catalyzed hydration of 2-trans-hexadecenoyl-CoA, was found to be a strong competitive inhibitor of the enzyme with a Ki of 0.35 microM. In contrast the short-chain product, L-3-hydroxybutyryl-CoA, is a weak competitive inhibitor with a Ki of 37 microM. L-3-Hydroxyhexadecanoyl-CoA is a much stronger inhibitor of crotonase than are other short-chain and long-chain intermediates of beta-oxidation and crotonase is more severely inhibited by this compound than are all beta-oxidation enzymes tested so far. Determination of true kinetic parameters for the crotonase-catalyzed hydration of long-chain substrates requires the removal of product in a coupled assay. When this was done, the Km for 2-trans-hexadecenoyl-CoA with bovine liver crotonase was found to be only 9 microM. It is suggested that under conditions of restricted beta-oxidation, when 3-hydroxyacyl-CoAs accumulate in mitochondria, the inhibition of crotonase by long-chain 3-hydroxyacyl-CoAs may limit the further degradation of medium-chain and short-chain intermediates of beta-oxidation.