Adenosine is present in the mammalian brain in large amounts and has patent effects on neuronal activity, but its role in neural signaling is poorly understood. The glutamate receptor agonist N-methyl-D-aspartate (NMDA) caused a presynaptic depression of excitatory synaptic transmission in the CA1 region of guinea pig hippocampal slices. This depression was blocked by an adenosine A1 receptor antagonist, which suggests that activation of the NMDA subtype of glutamate receptor raises the concentration of extracellular adenosine, which acts on presynaptic inhibitory A1 receptors. Strong tetanic stimulation caused a heterosynaptic inhibition that was blocked by both NMDA and A1 receptor antagonists. Enkephalin, which selectively inhibits interneurons, antagonized the heterosynaptic inhibition. These findings suggest that synaptically released glutamate activates NMDA receptors, which in turn releases adenosine, at least in part from interneurons, that acts at a distance to inhibit presynaptically the release of glutamate from excitatory synapses. Thus, interneurons may mediate a widespread purinergic presynaptic inhibition.