We investigated the spinal cords of 15 patients with sporadic amyotrophic lateral sclerosis (ALS) immunohistochemically using an anti-human neuronal nitric oxide synthase (nNOS) antibody to examine whether there is increased nNOS immunoreactivity in anterior horn neurons. Specimens from 16 patients without any neurological disease served as controls. In the controls, nNOS immunoreactivity of large anterior horn neurons was detected in 10 out of 16 cases. However, there were few nNOS-positive neurons, and most of large anterior horn neurons were spared. In the ALS patients, the mean number of nNOS-positive anterior horn neurons per transverse section of L4 and L5 was significantly larger (16.2 +/- 10.9) than that in the controls (7.0 +/- 9.2) (P < 0.0001). Moreover, 41.4% of large anterior horn neurons in ALS showed nNOS immunoreactivity in remarkable contrast to 7.6% in the controls. All ALS patients, whether showing mild, moderate or severe depletion of anterior horn neurons, displayed a higher percentage of nNOS-positive anterior horn neurons than the control patients showing nNOS immunoreactivity (P < 0.01). Most of the remaining anterior horn neurons in ALS showed more intense nNOS immunoreactivity on the surface of the neurons and their neuronal processes compared with the controls. Degenerated anterior horn neurons frequently demonstrated more intense nNOS immunoreactivity on the surface of the neurons than normal-appearing neurons. Some anterior horn cells displayed nNOS immunoreactivity in the somata. Dot-like nNOS deposits on anterior horn neurons were also positively immunoreactive with anti-synaptophysin antibody. Thus, increased nNOS expression is located mainly at the synaptic sites on the anterior horn neurons in sporadic ALS, which may be related to the degeneration of anterior horn neurons in this disease. Further studies are needed to determine whether the increased nNOS immunoreactivity plays a neuroprotective or neurotoxic role in the anterior horn neurons, and to show nitric oxide production in ALS.