Evidence for glutamate spillover in the hippocampus at physiological temperature remains controversial. We recorded from CA1 pyramidal cells (whole-cell mode) in acute hippocampal slices (obtained from young adult rats killed by decapitation) at 35 °C. We examined synaptic events mediated by NMDA receptors (NMDARs) by holding cells at +40 mV in the presence of CNQX. We divided stratum radiatum into two separate pathways that converged on the same cell but could be stimulated independently. After obtaining stable responses to single stimuli at each pathway, we applied the use-dependent NMDAR blocker MK801 and continued stimulation of only one (test) pathway until the EPSCs decreased to ~15 % of baseline. When we resumed stimulation of the other (silent) pathway, its responses were reduced to 48 ± 7 % (mean ± S.E.M.) of baseline (n = 7). This reduction could not be explained by the spontaneous activation of NMDARs in the presence of MK801: the latter reduced EPSCs only to 71 ± 6 % of baseline in control experiments (n = 15). Nor could it be explained by the activity-dependent changes in the occupancy of the NMDAR glycine-binding sites: 100 µM D-serine had no effect on the MK801-dependent reduction of EPSCs (n = 6). However, the MK801-dependent block of NMDARs at the silent pathway was exacerbated ~4-fold when glutamate uptake was blocked (n = 7), and ~2-fold when five stimuli at 50 Hz were applied to the test pathway (n = 8). Assuming that MK801 blocks the proportion p of NMDAR channels upon each stimulus, the normalised amplitude of NMDAR EPSCs at the nth stimulus is (1 – p)ⁿ. Our data then indicate that, on average, ~25 % of NMDARs at pyramidal cells can be activated by glutamate released at more than one synapse supplied by stratum radiatum. We also simulated extracellular glutamate diffusion in a 3D synaptic scatter compatible with neuropil in area CA1: the results provided quantitative insights into the relationship between synaptic activation and the extent of spatial glutamate ‘pools’ in the extracellular space.