The tissue inhibitors of metalloproteinases (TIMPs) are important regulators of the matrix metalloproteinases (MMPs), proteolytic enzymes essential for controlling the coordinated tissue remodeling that takes place in the ovary. In the present study, we characterized the ovarian expression pattern of TIMP-4. The localization of TIMP-4 mRNA was determined by in situ hybridization in adult cycling rats. TIMP-4 mRNA on the day of estrus was expressed in a punctate pattern in stroma and in corpora lutea (CL) from previous cycles but not in newly formed CL or follicles. At metestrus, TIMP-4 mRNA was present in certain CL from the current and previous cycles and continued to exhibit a punctate pattern of expression in the stroma. By diestrus, TIMP-4 mRNA was detected in the thecal layer surrounding follicles, and a relatively high level of expression was observed in a punctate pattern within new and previous CL and in the stroma. TIMP-4 mRNA was also observed in the thecal layer at proestrus, but the punctate pattern within CL and stroma was absent. To correlate the changes in cellular localization with changes in overall TIMP-4 levels, ovarian mRNA and protein levels were examined in adult cycling rats and in gonadotropin-stimulated immature rats. In cycling rats, there was no change in mRNA or protein levels across the cycle, although there was a trend towards higher levels during estrus (P = 0.08). In gonadotropin-treated rats, there was an increase in TIMP-4 mRNA 48 h after eCG administration with a corresponding doubling of TIMP-4 protein. Although TIMP-4 mRNA and protein tended to decline after hCG treatment, this trend was not significant (P = 0.08). These findings indicate that TIMP-4 could play an important role in regulating MMPs in a localized manner in follicles and CL throughout the cycle.