Two types of cDNA, which code for the two types of rat pancreatic secretory trypsin inhibitors (PSTIs), were cloned and sequenced. Both predicted amino acid sequences consisting of 79 amino acids, with the secretion signal peptide consisting of 18 and 23 amino acids for PSTI-I and PSTI-II, respectively. The nucleotide sequences were 91% homologous between the two cDNAs, but 68% and 65% homologous, respectively, when compared with human PSTI cDNA. Northern blot analyses showed that PSTI-I is expressed in the pancreas, whereas PSTI-II is expressed in the pancreas and the liver using the same promoter. Southern blot analyses suggested that both PSTI-I and PSTI-II genes are single copy genes per haploid genome. Duplication of rat PSTI gene seems to have occurred recently, after the divergence of humans and rats.