The purpose of this study was to evaluate mammalian ovarian follicular fluid (FF) for the components of a thrombin generating/modulating pathway. It is postulated that thrombin has a role in ovarian cell function through the activation of its functional receptor. In bovine, equine, ovine and porcine FF, functional activity of prothrombin (PT), Factor V (FV), Factor VII (FVII), Factor X (FX) and antithrombin (AT) were determined. Comparison of this profile in equine FF obtained by transvaginal aspiration and at slaughter showed no significant difference (p > 0.05) in PT, FV, FVII, FX, or AT activity. Western blot analysis revealed that PT and AT antigens in FF exhibit similar molecular weight as plasma PT and AT. Further, thrombin can be generated within FF by both direct and indirect activation of PT. These results suggest that thrombin formation is a common feature of all mammalian FF suggesting a role for thrombin in folliculogenesis.