In order to determine the localization of the peroxisomal and the mitochondrial pathways of beta-oxidation in the rat kidney, fatty acyl-coenzyme A (CoA) oxidase and 3-hydroxyacyl-CoA dehydrogenase activities were measured in glomeruli and in eight proximal and distal segments of the nephron. Structurally defined segments were dissected and analyzed with microchemical assays. The peroxisomal fatty acyl-CoA oxidase is restricted to the proximal tubule. The 3-hydroxyacyl-CoA dehydrogenase activity represents mainly the mitochondrial pathway and is similarly distributed in all cortical proximal and distal segments. It is much lower in glomeruli and collecting ducts. The distribution patterns of the two enzymes remain the same after 48 hr of starvation, although the activity of fatty acyl-CoA oxidase increases in glomeruli, proximal convolution, and collecting ducts. It is concluded that the capacity for mitochondrial beta-oxidation of fatty acids is similar in the proximal and the distal nephron. The proximal tubule possesses, additionally, a peroxisomal pathway for beta-oxidation with a capacity of the same order of magnitude as in liver cells.