Regulation of colonic motility depends upon the integrity of enteric inhibitory neurotransmission mediated by nitric oxide (NO), purine neurotransmitters, and neuropeptides. Intramuscular interstitial cells of Cajal (ICC-IM) and platelet-derived growth factor receptor α-positive (PDGFRα⁺) cells are involved in generating responses to NO and purine neurotransmitters, respectively. Previous studies have suggested a decreased nitrergic and increased purinergic neurotransmission in Kit^W/Kit^W-v (W/W^v) mice that display lesions in ICC-IM along the gastrointestinal tract. However, contributions of NO to these phenotypes have not been evaluated. We utilized small-chamber superfusion assays and HPLC to measure the spontaneous and electrical field stimulation (EFS)-evoked release of nicotinamide adenine dinucleotide (NAD⁺)/ADP-ribose, uridine adenosine tetraphosphate (Up4A), adenosine 5'-triphosphate (ATP) and metabolites from the tunica muscularis of human, monkey and murine colons and circular muscle of monkey colon, and we tested drugs that modulate NO levels or blocked NO receptors. NO inhibited EFS-evoked release of purines in the colon via presynaptic neuromodulation. Colons from W/W^v, Nos1^-/- and Prkg1^-/- mice displayed augmented neural release of purines that was likely due to altered nitrergic neuromodulation. Colons from W/W^v mice demonstrated decreased nitrergic and increased purinergic relaxations in response to nerve stimulation. W/W^v mouse colons demonstrated reduced Nos1 expression and reduced NO release. Our results suggest that enhanced purinergic neurotransmission may compensate for the loss of nitrergic neurotransmission in muscles with partial loss of ICC. The interactions between nitrergic and purinergic neurotransmission in the colon provides novel insight into the role of neurotransmitters and effector cells in the neural regulation of gastrointestinal motility.