The gene structure, expression and enzyme activity of a serine protease from the firefly, Pyrocoelia rufa (PrSP) were examined. The PrSP gene spans 1474 bp and consists of two introns and three exons coding for 257 amino acid residues. Southern blot analysis of genomic DNA suggested the presence of PrSP gene as a single copy. Western blot analysis and enzyme activity assay exhibited midgut-specific expression, suggesting that the midgut is the prime site where large quantities of PrSP are synthesized for degrading the absorbed protein from the diet. The cDNA encoding PrSP was expressed as a 31 kDa polypeptide in the baculovirus-infected insect Sf9 cells and the recombinant PrSP showed activity in the protease enzyme assay using gelatin as a substrate.