Myenteric neurons which innervate the mucosa of the guinea-pig ileum were characterized by combining retrograde transport of DiI in vitro with immunohistochemistry. Of DiI-labelled myenteric neurons, 43% were immunoreactive for calbindin and substance P, 25% were immunoreactive for calbindin alone, and 18% were immunoreactive for substance P alone. These 3 classes of neurons had Dogiel Type II morphology and are probably sensory neurons. Two classes of probable secretomotor neurons were characterized by immunoreactivity for neuropeptide Y (4%) and vasoactive intestinal peptide (2%). These 5 classes of myenteric neurons represent over 90% of the retrogradely labelled myenteric neurons that project to the mucosa.