Functional evidence suggests that nitric oxide released from CA1 pyramidal cells can act as a retrograde messenger to mediate hippocampal long-term potentiation, but the failure to find neuronal nitric oxide synthase (NOS-I) in the dendritic spines of these cells has cast doubt on this suggestion. We hypothesized that NOS-I may be in spines but in a form inaccessible to antibody when using standard histological fixation procedures. Supporting this hypothesis, we found that after a weak fixation protocol shown previously to enhance staining of synaptic proteins, CA1 pyramidal cells exhibit clear immunoreactivity for NOS-I. Confocal microscopy revealed that numerous dendritic spines in the stratum radiatum contained the NR2 subunit of the NMDA receptor and the adaptor protein postsynaptic density-95, and a subset of these spines also contained NOS-I. Quantitative studies showed that only approximately 8% of synaptic puncta (identified by synaptophysin staining) were associated with NOS-I, and approximately 9% contained the beta subunit of soluble guanylyl cyclase (sGC), a major target of NO. However, the majority of NOS-I-positive synaptic puncta was associated with sGC and vice versa. Postembedding immunogold electron microscopy showed that NOS-I concentrates just inside the postsynaptic plasma membrane of asymmetric axospinous synapses in the stratum radiatum of CA1, whereas sGCbeta concentrates just inside the presynaptic membrane. Together, these findings support the possibility that NO may act as a retrograde messenger to help mediate homosynaptic plasticity in a subpopulation of synapses in the stratum radiatum of CA1.