Rat pancreatic secretory trypsin inhibitor-I (PSTI-I) and -II (PSTI-II) were expressed in Saccharomyces cerevisiae AH22 under the control of the PHO5 promoter. Both proteins were secreted into the culture broth and purified by affinity chromatography using a genetically engineered mutant Streptomyces fradiae trypsin-like enzyme as a ligand. Rat PSTI-I was purified to homogeneity by one-step affinity chromatography, but the PSTI-II fraction contained two molecular mass forms (6.2 kDa and 5.6 kDa) after affinity chromatography. N-terminal amino acid sequencing revealed that the larger molecular mass protein was intact rat PSTI-II and five amino acid residues at its N-terminal had been truncated to the smaller form. The N-terminal amino acid sequences, amino acid compositions and trypsin inhibitory activities of recombinant rat PSTI-I and -II were identical to those of natural PSTIs. Moreover, the pancreatic exocrine secretion activity of recombinant rat PSTI-I in vivo also agreed well with that of natural PSTI-I.