A linear rate of H2O2 production is found when glutaryl-CoA is incubated with liver homogenates. We term this enzyme activity glutaryl-CoA oxidase. Its main characteristics are described and compared with those of glutaryl-CoA dehydrogenase (EC 18.104.22.168) and palmitoyl-CoA oxidase (EC 1.1.3.-). The latter enzyme catalyses the first step of peroxisomal beta-oxidation. Glutaryl-CoA oxidase shares several properties with palmitoyl-CoA oxidase. The activities of both enzymes in mouse liver are increased by feeding the animals with a clofibrate-containing diet. Subcellular fractionation of the liver homogenates on a linear sucrose gradient indicates that glutaryl-CoA oxidase is a peroxisomal enzyme.