Nitric oxide synthase immunoreactivity (bNOS-ir) was examined in the sacral spinal cord of the cat, macaque monkey and human using an antibody to the c-terminal region of neuronal NOS. In S2 of all 3 species NOS-ir was identified in both dorsal and ventral horns. In cat, monkey and human, bNOS-r occurred in sensory neurons of superficial laminae and the base of the dorsal horn, in autonomic neurons around the central canal and in the intermediolateral sacral spinal nucleus. In all 3 species, a large proportion of somatic motor nuclei in the ventromedial (VM), ventrolateral (VL) nuclei, and Onuf's nucleus (ON) showed high bNOS-ir, while others exhibited markedly lower immunoreactivity. Validatory experiments showed separate cellular localisation for bNOS, inducible NOS (iNOS), and endothelial NOS (eNOS) with only bNOS being localised to neuronal perikarya. Comparative morphometric analyses of the relative proportions and diameters of motor neurons in the VL, VM and ON exhibiting high and low levels of bNOS-ir revealed statistically significant differences in proportions in individual nuclei, and differences in size were generally not statistically significant. Finally, a comparison between cat sacral and thoracic spinal cord showed bNOS-ir in motor neurons of S2 was subject to less animal and rostrocaudal segment variation than in T10.