Expression of vasoactive intestinal polypeptide has been shown, by immunocytochemistry and biochemical assay, to follow the craniocaudal neural colonization of the mammalian gut. The aim of this study was to use in situ hybridization to see if it could provide more information on vasoactive intestinal polypeptide gene expression in the developing human gut. Immunocytochemistry of vasoactive intestinal polypeptide and, to visualize the total innervation, protein gene product 9.5 was also applied. By 8 weeks of gestation, protein gene product 9.5-immunoreactive neurons had colonized the gut lengthwise (17% of intestinal muscle area) but not transversely. Vasoactive intestinal polypeptide immunoreactivity was first detected at 9 weeks of gestation in a few nerve fibers of the upper gut, the origin of which could not be determined. Vasoactive intestinal polypeptide-immunoreactive ganglion cells were not seen until 18 weeks of gestation, whereas in situ hybridization showed messenger RNA in ganglion cells of the upper gut at 9 weeks. An adultlike pattern of peptide gene products (e.g., 2.5% and 3.1% of intestinal mucosal or muscle area, respectively) was detected by 20 weeks' gestation. The finding that the vasoactive intestinal polypeptide gene is expressed first in the upper human gut is consistent with craniocaudal neuronal colonization and maturation.