The role of the glycine modulatory site in N-methyl-D-aspartate receptor function was examined by determining the effect of the glycine site antagonist, 7-chlorokynurenic acid, on the induction of long-term potentiation at the commissural-CA1 synapse in anesthetized rats. Robust long-term potentiation of population excitatory postsynaptic potentials and population spike responses recorded extracellularly in the stratum pyramidale and in stratum radiatum of CA1 developed after high frequency stimulation (100 Hz for 1 s) of commissural fibers during continuous intrahippocampal administration of vehicle solution (0.15 M NaCl). In contrast, infusion of either 7-chlorokynurenic acid (400 microM) or of the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonovaleric acid (100 microM), significantly attenuated or completely blocked the development of long-term potentiation. When 7-chlorokynurenic acid was infused together with the glycine analog, D-serine (1 mM), long-term potentiation developed that was comparable to that observed in control animals. Intrahippocampal administration of D-serine alone was associated with slightly greater magnitude of long-term potentiation than observed in control animals. Collectively, these findings establish that in intact hippocampus, activity at the glycine modulatory site is necessary for activation of the N-methyl-D-aspartate receptor complex. Furthermore, these results suggest that the glycine modulatory site may not be fully saturated in vivo, and thus can serve to regulate N-methyl-D-aspartate receptor function.