The kinetic properties of the purified trifunctional beta-oxidation complex (TOC) from pig heart mitochondria were analyzed with the aim of elucidating the functional consequence of having three sequentially acting enzymes of beta-oxidation associated in one complex. The kinetic parameters of TOC and of the component enzymes of TOC, long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-ketoacyl-CoA thiolase, were determined with substrates having acyl chains with 16 carbon atoms. Quantification by high performance liquid chromatography of intermediates formed during the degradation of 2-trans-hexadecanoyl-CoA to myristoyl-CoA and acetyl-CoA by TOC revealed the accumulation of 3-hydroxyhexadecanoyl-CoA, whereas 3-ketohexadecanoyl-CoA was undetectable. The observed rates of NADH and acetyl-CoA formation were higher than the theoretical rates calculated by use of the kinetic parameters and measured concentrations of intermediates. When the sequence of reactions catalyzed by TOC was inhibited by acetyl-CoA, the steady-state concentration of the 3-hydroxyacyl-CoA intermediate was not affected, whereas a small amount of 3-ketohexadecanoyl-CoA was detected. The differences between observed and predicted reaction rates and between measured and expected concentrations of intermediates are best explained by the operation of a channeling mechanism. As a consequence of intermediate channeling between the active sites on the complex, more coenzyme A is available in the mitochondrial matrix and metabolites like 3-ketoacyl-CoA thioesters, which are strong inhibitors of several beta-oxidation enzymes, do not accumulate.