As a step in elucidating the biological role of plasma kallikrein (PK) present in the follicular fluid of mammalian ovaries, we examined pig ovary fluid to determine its constituent activators and substrates. Using the inactive precursor form of plasma kallikrein (prePK) as a substrate, we purified an enzyme capable of activating this protein. The prePK-activating enzyme was shown to be the active enzyme blood coagulation factor XIIa. We also isolated high molecular weight kininogen (HMW-K) from the same fluid. Incubation of HMW-K with the ovarian follicular fluid PK resulted in the production of the nanopeptide bradykinin (BK). Expression of prePK, blood coagulation factor XII, and HMW-K was examined by Northern blot analysis using ovary and liver poly(A) RNA. All these transcripts were found in the liver, but none were found in the ovary. In addition, it was found that BK levels in the fluid derived from the small follicles were approximately 6 times higher than those from medium and large follicles. These results demonstrate the presence of a BK-producing system in the ovarian follicles and suggest the physiological importance of this peptide hormone in the early stages of follicular development and at ovulation.