Abstract–Mammalian brain contains 2N‐acylamino acid amidohydrolases resembling the respective kidney enzymes. The anatomical and subcellular distribution of the enzymes; the substrate specificities and interspecies activities of the partially purified amidohydrolases have been studied. A considerable proportion of the amidohydrolase activity of subcellular fractions was found to be associated with the paniculate matter. The increased activities of the particulate enzymes in the presence of Triton X‐100 and chloroacetyl amino acid substrates suggest that significant amounts of the enzymes are in the bound or latent