In females, reproductive activity relies on proper integration of daily and environmental changes as well as cyclic sex-steroid feedback. This study sought to investigate the role of the hypothalamic Arg-Phe amide-related peptide (RFRP)-3 in the daily and seasonal control of reproductive activity in female Syrian hamsters by analyzing the RFRP system and investigating the effects of central administration of RFRP-3 at different reproductive stages. In long day-adapted sexually active female hamsters, the number of c-Fos-activated RFRP immunoreactive neurons was reduced in the afternoon of diestrus and proestrus; the latter was correlated with increased kisspeptin activity and the luteinizing hormone (LH) surge. Moreover, acute RFRP-3 administration decreased LH secretion when given midafternoon, before the LH surge, and had no effect at other time points of proestrus or diestrus. These data indicate that RFRP-3 exerts a tonic inhibition on LH secretion, which is lifted at the time of the preovulatory surge on the afternoon of proestrus. In short day-adapted sexually inactive female hamsters, Rfrp expression is strongly inhibited in a sex steroid-independent manner, and prolonged central infusion of RFRP-3 completely reactivated the reproductive axis through increased kisspeptin expression, gonadotropin and estradiol secretion, and gonadal weight. These findings reveal a critical role of RFRP-3 in the control of reproductive activity in female rodents and suggest that RFRP neurons, acting alongside kisspeptin neurons, are essential for proper synchronization of reproductive activity with the time of the day, the stage of the estrous cycle, and the seasonal changes in photoperiod.