Response gene to complement 32 (Rgc32) has recently been suggested to be expressed in the ovary and regulated by RUNX1, a transcription factor in periovulatory follicles. In the present study, we determined the expression profile of the Rgc32 gene in the rodent ovary throughout the reproductive cycle and the regulatory mechanism(s) involved in Rgc32 expression during the periovulatory period. Northern blot and in situ hybridization analyses revealed the up-regulation of Rgc32 expression in periovulatory follicles. Rgc32 mRNA was also localized to newly forming corpora lutea (CL) and CL from previous estrous cycles. Further studies using hormonally induced luteal and luteolysis models revealed a transient increase in levels of Rgc32 mRNA at the time of functional regression of the CL. Next, the regulation of Rgc32 expression was investigated in vitro using rat preovulatory granulosa cells. The effect of human chorionic gonadotropin on Rgc32 expression was mimicked by forskolin, but not phorbol 12-myristate 13-acetate, and was mediated by the activation of progesterone receptors and the epidermal growth factor-signaling pathway. The mechanism by which RUNX1 regulates Rgc32 expression was investigated using chromatin immunoprecipitation and Rgc32 promoter-luciferase reporter assays. Data from these assays revealed direct binding of RUNX1 in the Rgc32 promoter region in vivo as well as the involvement of RUNX binding sites in the transactivation of the Rgc32 promoter in vitro. In summary, the present study demonstrated the spatial/temporal-specific expression of Rgc32 in the ovary, and provided evidence of LH-initiated and RUNX1-mediated expression of Rgc32 gene in luteinizing granulosa cells.