Long-term potentiation (LTP) in hippocampus is a type of synaptic plasticity that is thought to be involved in learning and memory. 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 hippocampal slices, but more rigorous tests are possible in dissociated cell culture. 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.