The hepatic beta-oxidation of 3-phenylpropionic acid (PPA) was studied by the use of subcellular fractions and purified enzymes with the aim of characterizing intermediates and the subcellular location of this pathway. Respiration measurements with coupled rat liver mitochondria indicate that PPA is efficiently metabolized by mitochondrial beta-oxidation. In contrast, the peroxisomal beta-oxidation of this compound is at best a very slow process, as evidenced by the low activity of peroxisomal acyl-CoA oxidase toward 3-phenylpropionyl-CoA. In mitochondria, 3-phenylpropionyl-CoA is effectively dehydrogenated to cinnamoyl-CoA, which is only slowly converted to benzoylacetyl-CoA due to the unfavorable equilibrium of the hydration of cinnamoyl-CoA to 3-hydroxy-3-phenylpropionyl-CoA. Benzoylacetyl-CoA is a substrate of 3-ketoacyl-CoA thiolase. The dehydration of 3-hydroxy-3-phenylpropionyl-CoA to cinnamoyl-CoA forms the basis for a sensitive and stereospecific assay of enoyl-CoA hydratases. The progress of this reaction, which proceeds to near completion, can be measured spectrophotometrically at 308 nm. Soluble mitochondrial and peroxisomal enoyl-CoA hydratases only act on the (R,L) isomer, whereas the peroxisomal D-3-hydroxyacyl-CoA dehydratase is specific for the (S,D) isomer. Both substrates can be easily prepared from the commercially available enantiomeric acids. It is concluded that PPA, a key compound in Knopp's classical study that led him to formulate the principle of beta-oxidation, is overwhelmingly, if not completely, degraded by mitochondrial beta-oxidation.