Previous studies have demonstrated that plasminogen activator (PA) activity is significantly high in semen of infertile men, which is also high in semen when azoospermia or oligozoospermia is induced by injection of testosterone enanthate (TE) into healthy adult men or rhesus monkeys. To further clarify the source and possible role of PA in semen, the present study was undertaken to examine: (1) whether the mRNAs for tissue type PA (tPA), urokinase type PA (uPA), and PA inhibitor-1 (PAI-1) are expressed in epididymis, seminal vesicle and prostate gland of rhesus monkeys; and (2) whether PA has some effect on in vitro sperm capacitation as judged by the potential of sperm motility, acrosome reaction (AR) and in vitro fertilization. Our results showed that (1) mRNAs for PA and PAI-1 were expressed in epididymis, seminal vesicle and prostate gland, and (2) uPA, but not tPA, improved sperm mobility, induced AR and enhanced sperm capacity to fertilize mature eggs. Thus, it is concluded that PA activity in semen comes not only from testis and epididymis, but also from seminal vesicle and prostate gland; and that uPA, but not tPA, may play a role in sperm capacitation.