BACKGROUND & AIMS: Gastrointestinal motility persists when peripheral cholinergic signaling is blocked genetically or pharmacologically, and a recent study suggests nitric oxide drives propagating neurogenic contractions. METHODS: To determine the neuronal substrates that underlie these contractions, we measured contractile-associated movements together with calcium responses of cholinergic or nitrergic myenteric neurons in unparalyzed ex vivo preparations of whole mouse colon. We chose to look at these 2 subpopulations because they encompass nearly all myenteric neurons. RESULTS: Many, but not all, cholinergic neurons of the middle colon exhibited contractile-associated calcium responses with distinct features. By contrast, a large population of nitrergic neurons of the middle colon shut their activity off just before contraction onset, whereas another population of nitrergic neurons initiated a response just after contraction onset. When contractions were evoked by a variety of stimuli to the proximal and distal colon, the same neuronal subtypes exhibited the same activity patterns during the contraction. However, stimulation of proximal colon produced a transient, stimulation-locked response before the ensuing contraction in a subpopulation of cholinergic neurons and in nearly all nitrergic neurons, suggesting that distinct neuronal activity patterns underlie specific stimuli. Finally, although blockade of nitric oxide failed to arrest the generation or propagation of neurogenic contractions, chemogenetic elimination of nitrergic activity impaired their propagation to middle and distal colon. CONCLUSIONS: Genetic approaches were used to study the activity patterns of enteric neurons underlying spontaneous and evoked neurogenic contractions in unparalyzed colon. These approaches can be combined with a variety of other approaches to identify the neuronal subtypes and subclasses that coordinate colonic motility.