Tissue inhibitor of metalloproteinase (TIMP)-1 is a multifunctional peptide that has been implicated in the ovulatory process. To assess the function of TIMP-1 during the periovulatory period in vivo, mice incapable of expressing the TIMP-1 gene product were utilized. Twenty-three-day-old TIMP-1-deficient (n = 59) and wild-type (n = 61) female mice were injected with 5 IU eCG, followed 48 h later by an ovulation-inducing dose of hCG (5 IU). Animals were killed at the time of hCG injection (0-h hCG), at 12 h (12-h hCG), or at 24 h post-hCG (24-h hCG) administration. Serum was collected for the assessment of estradiol-17beta (0-h hCG groups) or progesterone content (12- and 24-h hCG groups), while ovaries were removed for either histological preparation or Northern analysis of TIMP-1, TIMP-2, and TIMP-3. The number of healthy and atretic follicles was determined in the 0-h hCG groups, as was the number of oocytes released in the 24-h hCG group. TIMP-1-deficient females in the 0-h hCG group showed reduced levels of ovarian TIMP-2 (0.29-fold decrease, p < 0.05) and TIMP-3 (3.0-fold decrease, p < 0.05) expression compared to wild-type counterparts. No significant difference was detected between genotypes in the 0-h hCG group for number of healthy or atretic follicles or for serum estradiol-17beta concentrations. Additionally, no significant differences were detected between genotypes in the 12- and 24-h hCG groups for serum progesterone concentrations, ovarian TIMP-2 and TIMP-3 expression, or number of oocytes released (24-h hCG group). To assess the effect of TIMP-1 on steroidogenesis in vitro, granulosa cells were obtained from 23-day-old, eCG-primed TIMP-1-deficient and wild-type females. Addition of recombinant human TIMP-1 significantly increased conditioned media estradiol-17beta concentrations in cell cultures from both mutant (1.32-fold over controls; p = 0.02; n = 4) and wild-type females (1.16-fold over controls; p = 0.04; n = 3). It is concluded from this study that TIMP-1 may modulate ovarian TIMP-2 and TIMP-3 mRNA expression during folliculogenesis. In addition, TIMP-1 exhibits steroidogenic activity in vitro, but no evidence was found for regulation of steroidogenesis in vivo.