We have constructed a recombinant baculovirus expression vector containing rat pancreatic lithostatin cDNA. Baculovirus infection of Spodoptera frugiperda (sf9) insect cells resulted in the de novo synthesis and secretion of a recombinant protein demonstrating an apparent molecular weight of about 16.5 kDa. Under optimal conditions [multiplicity of infection of 5 plaque-forming units (pfu)/cell and culture times of 48-56 h postinfection] recombinant protein was secreted into the culture medium at 5-10 mg/L. The secretory form of the recombinant protein was judged to be rat pancreatic lithostatin by the following criteria: (a) Trypsin cleavage resulted in limited proteolysis of the secreted product giving rise to a trypsin-resistant 15.5-kDa peptide, consistent with the size of the "pancreatic stone/thread protein"; (b) polyclonal antibodies raised against the recombinant protein identified 16.5-kDa secretory proteins in both rat pancreatic juice and sf9 culture medium; and (c) immunohistochemistry indicated that the native antigen resides within zymogen granules in pancreatic acinar cells.