Physiological studies on functionally identified myenteric neurons are scarce because of technical limitations. We combined retrograde labeling, cell culturing, and fluorescent intracellular Ca²⁺ concentration ([Ca²⁺]_i) signaling to study excitatory neurotransmitter responsiveness of myenteric motor neurons. 1,1-Didodecyl-3,3,3',3'-tetramethyl indocarbocyanine (DiI) was used to label circular muscle motor neurons of the guinea pig ileum. DiI-labeled neurons were easily detectable in cultures prepared from these segments. The excitatory neurotransmitters (10⁵ M) acetylcholine, substance P, and serotonin induced a transient rise in [Ca²⁺]_i in subsets of DiI-labeled neurons (66.7, 56.5, and 84.3%, respectively). DiI-labeled motor neurons were either inhibitory (23.8%) or excitatory (76.2%) as assessed by staining for nitric oxide synthase or choline acetyltransferase. Compared with excitatory motor neurons, significantly fewer inhibitory neurons in culture responded to acetylcholine (0 vs. 69%) and substance P (12.5 vs. 69.2%). We conclude that combining retrograde labeling and Ca²⁺ imaging allows identification of differential receptor expression in functionally identified neurons in culture.