Conditioning stimulation applied to afferent fibers in stratum radiatum or stratum oriens of hippocampal area CA1 produced heterosynaptic, posttetanic depression (PTD) of excitatory postsynaptic potentials (EPSPs). PTD amounted to a 60-80% reduction of EPSPs and recovered over a 5 min period. Conditioning stimulation also induced a posttetanic hyperpolarization (PTH) averaging 4 mV and decaying over a 1-1.5 min period. PTH was accompanied by a large reduction in input resistance. We sought to determine the pre- or postsynaptic locus of heterosynaptic PTD. Our results suggest that PTD reflects a presynaptic mechanism: (1) PTD was observed for both N-methyl-D-aspartate (NMDA) and non-NMDA receptor mediated EPSPs; (2) Direct depolarization of pyramidal cells, substituted for the synaptic depolarization induced by conditioning stimulation, did not elicit PTD; (3) PTD and PTH were differentially affected by pharmacological and postsynaptic manipulations; (4) Conditioning stimulation depressed responses to pressure applied glutamate, but the magnitude and duration were too small to account for PTD. Since afferent fiber volleys were not depressed following conditioning stimulation, while field EPSPs were, we conclude that conditioning stimulation suppresses synaptic release of glutamate.