Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyric acid-L-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa = beta Ala (carnosine, K(m) 1.2 mM), N-methyl beta Ala, Ala, Gly, and gamma-aminobutyric acid (homocarnosine, K(m) 200 microM), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn(2+) for full activity, and is sensitive to inhibition by bestatin (IC(50) 7 nM). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC ), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC ).