Guanine nucleotides have been examined for their effect on [3H]1-[1-(2-thienyl)-cyclohexyl]-piperidine ([3H]TCP) binding to rat forebrain synaptic plasma membranes (SPM). We report that of the series of guanine nucleotides tested, GTP, GDP, 5'-guanylylimidodiphosphate (Gpp(NH)p) and 5'-guanylylmethylenediphosphate (Gpp(CH2)p) are significantly more potent at decreasing [3H]TCP binding than GMP, cyclic GMP, and guanosine. GTP, the most potent compound tested, inhibited basal [3H]TCP binding with an IC50 of 38.7 microM. Stimulation of [3H]TCP binding with either the N-methyl-D-aspartate (NMDA) agonist, L-glutamate, or Mg2+ was also inhibited by GTP. Addition of GTP resulted in a rightward shift in the glutamate dose-response curve and a decrease in the maximum level of stimulation. The Mg2+ stimulation of [3H]TCP binding was completely blocked by the addition of GTP. These results, coupled with the previous findings that guanine nucleotides inhibit the binding of L-[3H]glutamate to the NMDA recognition site (Monahan et al., 1988), indicate that guanine nucleotides antagonize NMDA receptor-mediated neurotransmission, at least in part, through their action (direct or indirect) on the NMDA recognition site and thus may be endogenous negative modulators of the NMDA receptor.