The retrograde tracer fluoro-gold was injected into the periaqueductal grey, thalamus or hypothalamus, and spinal cord sections were processed for neuronal nitric oxide synthase (nNOS) immunohistochemistry to investigate the relationships of nNOS immunoreactive, and spinomesencephalic, spinothalamic and spinohypothalamic projection neurones. In addition, in the lateral spinal nucleus the relationship between spinomesencephalic, -thalamic and -hypothalamic projection neurones, and nNOS and interferon-gamma receptor immunoreactive structures was investigated at the lumbar level. No single retrogradely labelled spinomesencephalic, -thalamic or -hypothalamic neurone showed nNOS immunoreactivity. In the lateral spinal nucleus, however, many fluoro-gold-labelled neurones were closely apposed by both nNOS and interferon-gamma receptor immunoreactive structures, especially prominent in the hypothalamic injection cases. This study gave no evidence for nNOS immunoreactivity in spinal neurones projecting to the periaqueductal grey, thalamus or hypothalamus, but suggests that in the lateral spinal nucleus such neurones are contacted by both nNOS- and interferon-gamma receptor-containing axon terminals.