A dense plexus of fibers in the substantia gelatinosa contains nitric oxide synthase (NOS). Using electron microscopic double-labeling immunocytochemistry for NOS combined with GABA or glutamate, we find that all NOS-positive terminals in this region also contain GABA but are not enriched in glutamate. In an attempt to verify that NOS-positive terminals do not originate from primary afferents, we combined NOS immunocytochemistry with anterograde tracing from the sciatic nerve. An intrinsic spinal origin for the NOS-positive plexus is suggested. The results are discussed in the light of the possible involvement of nitric oxide in hyperalgesia.