A new member of the carboxypeptidase gene family, carboxypeptidase A2 (CPA2), has been identified from the predicted amino acid sequence of a rat pancreatic cDNA clone. In vivo recombination and in situ hybridization techniques employing the CPA2 cDNA resulted in the isolation of two genomic clones spanning the 25-kilobase pair rat CPA2 gene. Evolutionary trees built from the amino acid sequences of the known pancreatic carboxypeptidases show that CPA2 and carboxypeptidase A1 (CPA1) are the products of genes which duplicated before the mammalian radiation, and that bovine CPA is of the A1 type. The substrate specificities of CPA1 and CPA2 isolated from rat pancreas are similar to bovine CPA in that carboxyl-terminal amino acids with aromatic or branched aliphatic side chains are preferred. However, the substrate preference of rat CPA1 is skewed toward smaller amino acids, while that of rat CPA2 is skewed toward bulkier amino acids as compared to bovine CPA. The differences in the substrate specificities of these three carboxypeptidases are compatible with the nature of the amino acid replacements in their binding pockets for the carboxylterminal amino acid of the substrate.