Several lines of evidence suggest reproduction in the ranid frogs is potently regulated by the gonadal steroids, in particular 5alpha-dihydrotestosterone (DHT) and 17beta-estradiol (E(2)), and a non-gonadal steroid, the stress hormone corticosterone (Cort). Little is known about how these steroid hormones act upon the GnRH system to regulate the downstream reproductive events. We address these gaps in our knowledge by investigating the effects of Cort, E(2), and DHT administration on the in vitro release of GnRH and on the spermatogenesis of adult male leopard frog, Rana pipiens. R. pipiens were implanted for 20 days with silastic capsules containing cholesterol (Ch; control), Cort, E(2), or DHT. Upon sacrifice, acute hypothalamic explants were cultured and measured for GnRH release, and testes processed for histological analysis. Although only E(2) implant significantly reduced the gonadosomatic index, all three steroid hormones altered spermatogenesis. Cort modestly but significantly reduced the presence of spermatids. The effects of E(2) and DHT were both stimulatory and inhibitory, depending on the stage of spermatogenesis. None of the steroid hormones altered baseline GnRH release. Interestingly, only E(2) significantly stimulated veratridine-induced GnRH release, suggesting E(2) treatment increased the releasable pool of GnRH and/or enhance the excitability of GnRH neurons. In sum, this is the first study to report the direct measurement of GnRH secretion in a poikilothermic tetrapod. Our results revealed potent but sometimes paradoxical effects of steroid hormones, especially E(2), on the reproductive regulation of the male R. pipiens.