The properties and subcellular localization of fatty acyl-CoA oxidase (FAO) were studied in rat heart homogenates. After differential centrifugation, FAO was sedimentable and enriched in a “light-mitochondrial” fraction. FAO had a pH optimum of 8–9. Among straight-chain, saturated fatty acyl-CoAs, the enzyme showed a marked preference for medium chain substrates (C12 > C10 = C8 > C16 = C14 > C6) over a concentration range up to 100 µM. No activity was observed with C4-CoA. The apparent Michaelis constant (Km) for C12-CoA was 5-10 µM. After removal of nuclei by low-speed centrifugation, combined subcellular particle preparations were obtained by high-speed centrifugation and layered on linear density gradients of metrizamide. After density equilibration, FAO showed a symmetric distribution centered at ρ = 1.16–1.18, like that of the enzyme catalase, a marker for microperoxisomes. In contrast, enzyme markers for mitochondria, lysosomes, sarcolemma, and sarcoplasmic reticulum were recovered in low-density regions of the gradient. These results provide a direct demonstration of fatty acyl-CoA oxidase in cardiac tissue and its association with microperoxisomes.