LONG–TERM potentiation (LTP) in hippocampus is a type of synap-tic plasticity that is thought to be involved in learning and memory¹. Several lines of evidence suggest that LTP involves 3′,5′-cyclic GMP (cGMP), perhaps as an activity-dependent presynaptic effector of one or more retrograde messengers (refs 2-12, but see ref. 13). However, previous results are also consistent with postsynaptic effects of cGMP. This is difficult to test in hippocam-pal slices, but more rigorous tests are possible in dissociated cell culture^14–17. We have therefore developed a reliable method for producing N-methyl-D-aspartate (NMDA) receptor-dependent LTP at synapses between individual hippocampal pyramidal neurons in culture. We report that inhibitors of guanylyl cyclase or of cGMP-dependent protein kinase block potentiation by either tetanic stimulation or low-frequency stimulation paired with postsynaptic depolarization. Conversely, application of 8-Br-cGMP to the bath or injection of cGMP into the presynaptic neuron produces activity-dependent long-lasting potentiation. The potentiation by cGMP involves an increase in transmitter release that is in part independent of changes in the presynaptic action potential. These results support a presynaptic role for cGMP in LTP.