We investigated the subcellular distribution of microsomal aldehyde dehydrogenase (msALDH) in rat liver and revealed by the immunoblotting method that msALDH or a cross-reacting 54-kDa protein(s) exists in the outer mitochondrial membranes and peroxisomes. Anti-msALDH antibody markedly inhibited the decanal aldehyde dehydrogenase activity of the outer mitochondrial membranes as well as that of the microsomes. Immunogold electron microscopic observations showed that gold particles are localized over the ER, outer mitochondrial membranes and peroxisomal membranes. These results suggest that msALDH or its cross-reacting related protein is distributed not only in the ER membranes but also in the mitochondrial outer membranes and peroxisomal membranes.