Since the serine protease inhibitor, protein C inhibitor (PCI), is present in seminal plasma at ≈3 μM, complexes of PCI with urokinase (uPA) and tissue type (tPA) plasminogen activator were quantitated using sandwich enzyme-linked immunosorbent assays (ELISA’s). Seminal plasma (N = 10) collected in the absence of extrinsic inhibitors had a mean of 25 ± 5 ng/ml uPA: PCI, 76 ± 23 ng/ml tPA: PCI, and 4 ± 2 ng/ml of tPA complexes with plasminogen activator inhibitor-1 (tPA:PAI-l). 93% of the uPA and 17% of the tPA antigen in seminal plasma was in complex with PCI and, when complexation was inhibited by collecting semen into an 1,10-phenanthrolinium solution, 33% of the uPA and 7% of the tPA was complexed to PCI. Urine (N = 10) contained 4 ± 1 ng/ml uPA:PCI. In purified system, complexation of uPA and tPA to PCI paralleled the inhibition of the enzymes. In vitro studies in blood and seminal plasma showed that heparin stimulated complexation of uPA and tPA with PCI, suggesting that negatively charged glycosaminoglycans in blood vessels and in the reproductive system may regulate PCI reactions with uPA and tPA. These results suggest that PCI is a physiologic regulator of uPA and tPA in male reproductive tissues and raises questions about a potential role of PCI in human fertility and in uPA-dependent cell invasiveness.