Rat carboxypeptidase A cDNA clones have been isolated from a cDNA library prepared from pancreatic mRNA. An almost complete mRNA sequence has been deduced that predicts a polypeptide having 78% amino acid sequence homology with bovine carboxypeptidase A. The amino acid sequence of the activation and signal peptides of the carboxypeptidase A precursor were inferred from the nucleotide sequence. The cDNA was used as a probe to identify DNA fragments containing carboxypeptidase A sequences in a bacteriophage lambda library of rat genomic DNA. Heteroduplexes revealed that the DNA coding sequence occupies 5.5 kilobases and is interrupted by nine intervening sequences. The nucleotide sequence of the 5' end of the gene and the adjacent flanking region provides information on the site of initiation of transcription and the putative control regions. There is no evident relationship between the localization of intervening sequences in the gene and functional/structural domains of the protein.