The involvement of N-methyl-D-aspartate (NMDA) receptors in synaptic transmission in the Schaffer collateral-commissural pathway of rat hippocampal slices has been examined in the presence and absence of Mg2+. Superfusion of slices with Mg2+ -free medium resulted in an increase in the amplitude of the population spike and the appearance of multiple population spikes. An NMDA antagonist, which had no effect on the synaptic response in the presence of Mg2+, completely prevented the appearance of multiple responses. If added to slices already bathed in Mg2+ -free medium, the antagonist eliminated the multiple responses and partly reduced the amplitude of the initial population spike. These data suggest that the extent to which NMDA receptors participate in synaptic transmission in rat hippocampus depends on the extracellular Mg2+ concentration.