By means of subcellular fractionation D-aspartate oxidase was shown to be localized in peroxisomes in rat and human liver. The oxidase from both sources was most active on D-aspartate and N-methyl-D-aspartate. In different rat tissues, the highest enzyme activity was found in kidney, followed by liver and brain. In these tissues, oxidase activities became detectable 1-4 days after birth, reaching adult values after 4 weeks. Analysis of liver samples from patients with Zellweger syndrome, a generalized peroxisomal dysfunction, demonstrated no significant deficiency of this particular oxidase.