The expression of the constitutive neural isoform of nitric oxide synthase (bNOS) is dynamic and thus forms an ideal parameter to evaluate whether development and region affect the enteric nervous system. By applying NADPH-diaphorase histochemistry on whole-mount preparations of the myenteric and submucosal plexuses and by using the 'unbiased counting frame', a qualitative and quantitative description of bNOS-expression in enteric neurons in the pig duodenum in various developmental stage and region was obtained. Examinations were carried out on the oral and aboral duodenum of fetal pigs from the second half of gestation, of 1-2-day-old pigs and of 6-8-week-old pigs. In the pig duodenum, three enteric plexuses were readily distinguished: the inner submucous, the outer submucous and the myenteric plexuses. All three plexuses already harboured, to different degrees, bNOS-expressing neurons at midgestation. Although the enteric nervous system was present at midgestation, the enteric neurons had not yet reached their adult phenotype and morphology. During gestation, the number of inner submucous bNOS-expressing neurons increased approximately 50-fold, whereas after birth that number fell to about 10% of the prenatal value. During further postnatal development it returned to prenatal values. In addition, the number of bNOS-expressing myenteric neurons doubled postnatally. These changes favour a role for NO in mediating the development of enteric neurons and point to a greater necessity for inhibitory innervation in the adult pig as compared with the fetal pig. Furthermore, the number of bNOS-expressing outer submucosal and myenteric neurons was significantly higher in the oral duodenal segment compared with the aboral duodenal segment. This regional difference suggests that the oral duodenal segment is more prominently involved in the regulation of NO-mediated gastrointestinal processes than the aboral one. The developmentally and regionally dependent bNOS-expression can be explained by shifts and differences in the balanced system of hormones, presynaptic input and target-derived signals that affects neurotransmitter expression.