D-Aspartate oxidase (EC 1.4.3.1; D-ASPOX) specifically oxidizes the D-isomers of dicarboxylic amino acids such as aspartic or glutamic acid. Subcellular fractionation experiments in the past showed its association with peroxisome preparations in kidney cortex and liver. However, no information exists on the in situ localization and distribution of the enzyme in different cell types. We have purified the enzyme from the bovine kidney and raised an antibody against it in rabbits. The monospecificity of the antibody has been confirmed by Western blotting and it does not crossreact with D-amino, acid oxidase. Immunohistochemical localization of the antigen in bovine kidney and liver with the streptavidin-biotin-peroxidase technique revealed a punctate localization in the epithelial cells of proximal nephron tubules, particularly in the straight P-3 segment, as well as in hepatocytes. This is consistent with a localization in peroxisomes. Best results have been obtained with Carnoy-fixed material after paraffin embedding or after fixation with formaldehyde-glutaraldehyde in cryostat sections. Immunoelectron microscopy with protein A-gold confirms the peroxisomal localization of D-ASPOX. Gold particles are distributed over the matrix, suggestive of a peroxisomal matrix enzyme. This is the first report on the localization of D-ASPOX, a little-known peroxisomal enzyme. The techniques described and the antibody prepared will now allow systematic investigation of its tissue distribution.