We have studied modulation of FSH-sensitive adenylate cyclase activity in testes of immature rats by guanyl nucleotides. Highly purified hFSH alone stimulated adenylate cyclase activity 2.2-fold over basal levels. Addition of the GTP analog, 5'-guanylyl imidodiphosphate [Gpp(NH)p], caused an additional 2.8-fold augmentation of adenylate cyclase activity to 6 times over basal levels and 3.7 times greater than that seen in the presence of Gpp(NH)p alone. GTP did not significantly stimulate basal levels of adenylate cyclase and augmented FSH stimulated activity by 1.4-fold; other nucleotides were without effect. Half-maximum activation of adenylate cyclase in each instance was produced by approximately similar concentrations of either guanyl nucleotide (about 10 microM). The Km for hormone activation of adenylate cyclase was nearly the same in the presence and absence of Gpp(NH)p. Maximum adenylate cyclase stimulation in the presence of nucleotide and/or hRSH was always less than obtained by fluoride alone. Of all nucleotides tested, only GTP and its analog, Gpp(NH)p, significantly augmented FSH stimulation of testicular adenylate cyclase activity. Gpp(NH)p also markedly inhibited binding of radiolabeled hFSH to testicular receptor, but at a concentration 15-fold greater than that required for significant stimulation of testicular adenylate cyclase activity. The results suggest a specific role for guanyl nucleoside triphosphate in regulation of FSH effects on testicular adenylate cyclase activity.