The gonadotropin surge is the essential trigger to stimulate ovulation and luteinization of ovarian follicles. While the hormone signals from the brain that initiate ovulation are known, the specific targets which regulate this process are not well known. In this study, we assessed the suitability of the Rhox homeobox gene cluster to serve as the master regulators of folliculogenesis. In superovulated (equine chorionic gonadotropin [eCG]/human chorionic gonadotropin [hCG]) mice, the Rhox genes exhibited four distinct windows of peak expression, suggesting that these genes may regulate specific events during the ovulatory cycle. Like many members of the cluster, Rhox8 mRNA and protein were induced by follicle stimulating hormone [FSH]/eCG in granulosa cells. However, Rhox8 displayed unique peak expression at 8 h post-hCG administration, implying it might be the lone member of the cluster regulated by progesterone. Subsequent promoter analysis in granulosa cells revealed relevant homeobox binding and progesterone response elements within Rhox8's 5′-flanking region. In superovulated mice, progesterone receptor (PGR) is recruited to the Rhox8 promoter, as assessed by chromatin immunoprecipitation. In Rhox5-null mice, Rhox8 mRNA was reduced at 2 h and 4 h post-hCG administration but recovered once the follicles passed the antral stage of development. Conversely, in progesterone receptor knockout mice, Rhox8 exhibited normal stimulation by eCG but failed to reach its peak mRNA level at 8 h post-hCG found in wild-type mice. This suggests a model in which Rhox8 transcription is dependent upon RHOX5 during early folliculogenesis and upon progesterone during the periovulatory window when RHOX5 normally wanes. In support of this model, transfection of RHOX5 and PGR expression plasmids stimulated, whereas dominant negative and mutant constructs inhibited, Rhox8 promoter activity.