The matrix metalloproteinases (MMPs) have been implicated in the ovulatory process, but the specific roles of individual MMPs are unclear. This study examined the effect of the preovulatory gonadotropin surge on localization and regulation of MMP-2, MMP-14, and tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA and MMP-2 and TIMP-2 activity in bovine preovulatory follicles and new corpora lutea (CL). Ovaries containing ovulatory follicles or new CL were collected at approximately 0, 6, 12, 18, 24, and 48 h (CL) after a GnRH-induced gonadotropin surge. Messenger RNA for TIMP-2 and MMP-14 increased within 6 and 24 h of the gonadotropin surge, respectively, whereas MMP-2 mRNA was constitutively expressed. Activity for MMP-2 in follicular fluid and follicle homogenates was not changed, but follicular fluid TIMP-2 activity increased in response to the gonadotropin surge. Messenger RNA for MMP-2 was localized to the thecal layer of bovine preovulatory follicles, whereas MMP-14 mRNA was localized primarily to the thecal layer and adjacent ovarian stroma. Expression of MMP-14 was also observed in the granulosal layer after the gonadotropin surge. In contrast, TIMP-2 mRNA was localized predominantly to the granulosal layer with intense expression in the antral portion of the granulosal layer in response to the gonadotropin surge. These data support the hypothesis that increased expression of MMP-14 and TIMP-2 may help regulate follicle rupture and/or the ovulatory follicle-CL transition in cattle.