Analogous to human thrombin, prophenoloxidase-activating proteinase (PA-P) is a terminal enzyme of a serine proteinase cascade in the tobacco hornworm Manduca sexta. In order to purify and study the activating enzyme for PAP from this insect, we produced the zymogen of PAP (proPAP) in a bacterial expression system. The affinity-purified protein was then used as an antigen to generate a specific rabbit antiserum. Immunoblot analysis indicated that the proPAP was present at a low level in Manduca larval hemolymph, but was induced by six- to eightfold in larvae that had been injected with Escherichia coli or Micrococcus lysodeikticus. To produce the native proenzyme for functional analyses, we constructed a recombinant baculovirus to infect Spodoptera frugiperda Sf21 cells. ProPAP was secreted into the medium at a low concentration of approximately 0.37 mg/liter under the optimal conditions. We then developed a simple, efficient scheme to enrich and purify this protein, which involves two lectin affinity and one HPLC ion-exchange chromatographic steps. Immunoblot analysis following SDS-polyacrylamide gel electrophoresis indicated that the recombinant proPAP is nearly identical in mobility to the zymogen from Manduca hemolymph. After the purified proPAP was added to the larval hemolymph, it was readily activated by an unknown proteinase in the presence of M. lysodeikticus.