GTP binding to Sertoli cell membranes has been investigated using [3H]5'-guanylyl-beta gamma-imidodiphosphate [[3H]Gpp(NH)p], a nonhydrolyzable analog of GTP. Binding of [3H]Gpp(NH)p Gpp(NH)p to membranes prepared from Sertoli cells in serum-free culture was proportional to membrane protein concentration in the range of 5-50 micrograms. Competitive displacement studies using adenine (ATP, ADP, and AMP) and guanine nucleotides [GTP, GDP, GMP, and Gpp(NH)p] indicated that only GTP, its analog Gpp(NH)p, and GDP were effective ligands. The relative potencies were Gpp(NH)p much greater than GTP greater than GDP, as characterized by ED50 values of 0.8, 2.5, and 4 microM, respectively. Competitive inhibition by GTP, however, was similar to that by Gpp(NH)p in the presence of a nucleoside triphosphate-regenerating system, suggesting the involvement of an active GTPase. Equilibrium binding studies indicated a single high affinity site for GTP with a Ka of 3.3 +/- 0.2 X 10(7) M-1. This value was supported by other studies in which an association rate constant of 1.8 X 10(6) M-1 min-1 and a dissociation rate constant of 2.4 X 10(-2) min-1 were estimated. Maximal binding of [3H]Gpp(NH)p to Sertoli cell membranes ranged from 30-55 pmol/mg protein. FSH enhanced [3H]Gpp(NH)p binding by about 50% (P less than 0.05), reflecting an increase in the number of available binding sites rather than an effect on Ka. When GDP was preincubated with membranes in the absence of FSH, the number of available binding sites for [3H]Gpp(NH)p was decreased. This reduction in available binding sites by pretreatment with GDP could be reversed by adding FSH during the equilibrium binding analysis. These studies have demonstrated specific high affinity binding of Gpp(NH)p to Sertoli cell membranes with an affinity comparable to that required for activation of FSH-sensitive adenylate cyclase. Furthermore, a potent GTPase activity associated with the Sertoli cell membrane is responsible for rapid hydrolysis of GTP to GDP and may participate in inactivation of GTP-dependent adenylate cyclase activity. The role of FSH in the regulation of nucleoside binding appears to be in facilitating exchange of GTP for GDP by enhancing the release of bound GDP.