This study was performed to clarify developmental changes in the structure of the myenteric nerve plexus in the rat small intestine. The small intestines of fetal and neonatal rats were examined by light microscopic immunohistochemistry for protein gene product 9.5 (PGP-9.5) and alpha-smooth muscle actin (alpha-SMA). Direct observation of the three-dimensional structure of the neuronal and muscular elements was accomplished by scanning electron microscopy of tissues after KOH treatment. At the embryonic day 14 (E14), PGP-9.5 immunopositive (neuronal) cells first appeared in the mesenchymal tissue outside an alpha-SMA positive (muscle) cell layer. These cells increased in number to form a thin and continuous sheet, which was sandwiched between the inner and outer muscular layers at E17. This neuronal layer consisted of immature ganglion cells associated with supportive (enteroglial) cells, and was gradually segregated into groups by incomplete separation through slit-like interruptions at E18. With the widening of these slits at E19, the neuronal sheet was observed as a ladder composed of transversely elongated ganglia and their connecting nerve strands, resulting in the typical structure of the myenteric nerve plexus. Fibroblastic cells of a peculiar shape appeared on the outer surface of the myenteric plexus at E19. These cells (presumably interstitial cells) interconnected their long branching projections to form a cellular network on the myenteric plexus until birth.