Classically, development of the human enteric nervous system has been characterized by the early appearance (between 9 and 12 weeks' gestation) of adrenergic and cholinergic nerves. The development of peptidergic innervation occurs much later. Recent studies have indicated that nitric oxide is involved in the nonadrenergic noncholinergic innervation of the gut, mediating its relaxation. The authors have investigated the ontogeny of nitrergic (nitric oxide synthase-containing) neurons of the developing gut. Bowel segments from the esophagus, pylorus, and ileocecal and rectosigmoid regions of 14 fetuses (gestational age range, 12 to 23 weeks) were studied with nicotinamide adenosine dinucleotide phosphate (NADPH) diaphorase histochemistry. By 12 weeks' gestation, nitrergic neurons had appeared in the myenteric ganglia, at all levels of the gut, and had begun plexus formation. Nitrergic innervation in the submucous plexus becomes evident after 14 weeks. As gestational age increases, nitrergic innervation becomes richer and more organized. Increasing numbers of nitrergic nerve fibers are seen in the circular muscle; some of these fibers project from the myenteric plexus. By 23 weeks' gestation, nitrergic innervation has matured to the pattern observed in the postnatal gut. Thus, the onset and pace of development of nitrergic innervation are similar to adrenergic and cholinergic innervation and occur before peptidergic innervation. This study provides morphological evidence of the ontogenetic significance of nitrergic innervation in the human gut and supports previous suggestions that nitric oxide has a pathophysiological role in developmental gut motility disorders.