Nitric oxide (NO) is involved in the inhibitory neurotransmission in the sphincteric and non-sphincteric smooth muscles. However, the relative contribution of nitric oxide synthesizing innervation to these functionally diverse parts of the gut, particularly during development, is unknown. Gastrointestinal sphincters and adjoining non-sphincteric bowel segments were obtained from 14 human fetuses with gestational ages between 12 and 23 weeks. NO synthesizing nerves were examined by nicotinamide adenine deoxinucleotide phosphate (NADPH) diaphorase histochemistry. The densities of NADPH positive nerves in the smooth muscle were quantified using a computerized image analyzing system on random sections. The NO synthesizing nerve density in intestinal smooth muscles decreased during fetal development as a result of increased interspacing between myenteric ganglia and a disproportionately larger increase in smooth muscle area than neuronal area. Similarly, the nerve densities were lower in sphincteric regions than adjoining non-sphincteric regions at the same gestational ages. There is a relative reduction of the density of NO synthesizing nerves in intestinal smooth muscle particularly in sphincteric regions during development. These findings may have relevance to the occurrence of congenital dysmotility disorders of the sphincteric regions.