The pattern of distribution and colocalization of nitric oxide synthase and the calcium-binding protein calretinin in myenteric neurons and nerve fibers were examined in the human small intestine from preterm fetuses (14-17 weeks of gestation), normal adults (mean age 50 years old), old age (mean age 80 years old), and Crohn's disease patients (mean age 30 years old) using NADPH-diaphorase histochemistry and immunohistochemical techniques. In all age groups investigated, NADPH-diaphorase-reactive and calretinin-immunoreactive neurons and nerve fibers were seen throughout the myenteric plexus. The highest proportion of NADPH-diaphorase-reactive neurons was found in the myenteric ganglia of old age intestines (56% of protein gene product-immunoreactive neurons) followed by fetal intestines (41%) and Crohn's intestine (30%) compared with intestines of control adults (20%). A similar trend was observed for calretinin-immunoreactive neurons where the highest proportion of immunoreactive neurons was found in the myenteric ganglia of old age intestines (28% of protein gene product-immunoreactive neurons), followed by fetal intestines (22%), and Crohn's intestines (18%) compared with intestines of control adults (9%). A colocalization of NADPH-diaphorase activity and calretinin immunoreactivity was only seen in the myenteric neurons of fetal intestines (2% of NADPH-diaphorase-reactive neurons were also calretinin-immunoreactive). The pattern of distribution of NADPH-reactive and calretinin-immunoreactive neurons in the myenteric ganglia of fetal intestine differs from that of the other age groups. In the fetal intestine, the myenteric neurons containing either calretinin or NADPH-diaphorase are distributed through out the myenteric ganglia with no specific orientation to one another. In the intestines of control adult, Crohn's, and old age patients, single large calretinin-immunoreactive neurons are surrounded by a number of small NADPH-diaphorase-positive neurons, with this feature being more prominent in intestines of old-age and Crohn's disease patients. In summary, a high number of both NADPH-diaphorase-reactive and calretinin-immunoreactive neurons were seen in the myenteric ganglia of fetal, old age, and Crohn's intestines; we discuss that there may be a role for nitric oxide and calretinin in the process of development, aging, and pathological changes in the human intestine associated with alteration in the calcium homeostasis in the myenteric neurons.