The process of ovulation involves weakening of the follicular wall by proteolytic enzymes. The function of FURIN (also known as PCSK3) is to activate various proteolytic enzymes. In the present study, the expression, localization, and function of FURIN were investigated in the periovulatory rat ovary. Immature female rats were injected with equine chorionic gonadotropin followed by human chorionic gonadotropin (hCG) 48 h later to stimulate ovulation. Ovaries were collected at 0, 4, 8, 12, and 24 h after hCG injection. Administration of hCG increased Furin mRNA expression in both intact ovaries and cultured ovarian follicles to maximal levels at 8 and 12 h before decreasing at 24 h. In cultured granulosa cells, Furin mRNA levels were significantly induced at 12 h after hCG. In situ hybridization of Furin mRNA demonstrated expression in the granulosa cells, with predominant expression in the theca layer. Regulation studies demonstrated that Furin mRNA was induced in residual tissue by forskolin or amphiregulin. To examine the role of FURIN in protease activation and ovulation, rats were treated with a FURIN inhibitor and oocyte release was determined. There was a 38% decrease in the number of oocytes released in ovaries treated with the FURIN inhibitor. Likewise, the FURIN inhibitor decreased the activation of MMP2. The induction of Furin mRNA after treatment with hCG, along with the decrease in MMP2 activation and oocyte release after FURIN inhibition, supports the hypothesis that FURIN is upregulated during the preovulatory period, which results in activation of proteinases associated with the breakdown of the follicular wall during ovulation.