The etiology of hypertrophic pyloric stenosis (HPS) is not known. We made an immunocytochemical examination of pyloric muscle from 18 patients with HPS and 10 controls using specific monoclonal antibodies to neural cell adhesion molecule (NCAM) as well as neurofilament protein and NADPH-diaphorase histochemistry. In HPS, bundles of hypertrophic muscle fibers expanded the circular muscle layer. The longitudinal muscle also appeared hypertrophic but to a less marked degree. The most striking difference between HPS and the control tissues was that NCAM, NADPH-diaphorase, and neurofilament protein immunoreactive fibers were absent or markedly reduced within the hypertrophied circular and longitudinal musculature. In contrast, NCAM, NADPH-diaphorase, and neurofilament protein immunoreactivity was preserved in the myenteric plexus where nerve fibers and ganglion cells were stained. The lack of expression of NCAM, NADPH-diaphorase, and neurofilament protein on nerve fibers within the circular and longitudinal muscle in patients with pyloric stenosis suggests that the smooth muscle is not innervated in this condition.