Background & Aims: Myenteric ganglia in the human gastrointestinal tract contain a mixture of many different types of nerve cells that cannot be distinguished by their location. The aim of this study was to characterize different functional types of cells by using retrograde labeling in vitro to identify neurons according to their targets. Methods: The retrograde label 1,1′-didodecyl 3,3,3′,3′-indocarbocyanine perchlorate (Dil) was applied to different target layers of human small or large intestine. After 3-5 days in organotypic culture, myenteric neurons projecting to the Dil application site were visualized and mapped using fluorescence microscopy. Results: Myenteric motor neurons projecting to the external muscle layer were typically unipolar cells with lamellar dendrites (Dogiel type I) and had short projections up to 16 mm long. In contrast, presumed intemeurons with Dogiel type I morphology were shown to project up to 68 mm aborally or up to 38 mm orally. Multipolar Dogiel type II neurons with smooth cell bodies were labeled most frequently from the submucous plexus. No myenteric neurons were labeled by Dil applied to the mucosa. Conclusions: Myenteric neurons labeled from each target had characteristic size, morphology, polarity, and length of projections, indicating that there is a high degree of organization in the human enteric nervous system.