Long-term potentiation, a persistent increase in synaptic efficacy, may require a retrograde signal originating in the postsynaptic cell that induces an increase in presynaptic neurotransmitter release. We have constructed a mouse homozygous for a targeted null mutation in the endothelial isoform of nitric oxide synthase and report that long-term potentiation in the CA1 region of these mice is entirely absent under weak stimulation conditions. Application of a membrane-permeant guanosine-3',5'-cyclic monophosphate analogue during tetanus fails to compensate for this deficit, suggesting that nitric oxide produced by endothelial nitric oxide synthase may affect long-term potentiation through a cascade that does not include guanylyl cyclase. We also report that strong tetanic stimulation can induce robust long-term potentiation in these mice which is not blocked by pharmacological inhibitors of nitric oxide synthase. Furthermore, mice lacking endothelial nitric oxide synthase show no shift in the frequency-response curve for the induction of long-term potentiation. Basal synaptic transmission, paired-pulse facilitation and the electrical properties of CA1 cells in these mice were similar to controls. These results support a selective role for endothelial nitric oxide synthase in long-term potentiation, but also demonstrate that nitric oxide synthase is not involved in this process under all conditions.