Nitric oxide (NO) is a major inhibitory neurotransmitter in the gastrointestinal (GI) tract. Its main effector, NO-sensitive guanylyl cyclase (NO-GC), is expressed in several GI cell types including smooth muscle cells (SMC), interstitial cells of Cajal (ICC) and fibroblast-like cells. Up to date the interplay between neurons and these cells to initiate a nitrergic inhibitory junction potential (IJP) is unclear. Here, we investigate the origin of the nitrergic IJP in murine fundus and colon. IJPs were determined in fundus and colon SMC of mice lacking NO-GC globally (GCKO) and specifically in SMC (SM-GCKO), ICC (ICC-GCKO) and both SMC/ICC (SM/ICC-GCKO). Nitrergic IJP was abolished in ICC-GCKO fundus and reduced in SM-GCKO fundus. In colon, the amplitude of nitrergic IJP was reduced in ICC-GCKO whereas nitrergic IJP in SM-GCKO was reduced in duration. These results were corroborated by loss of the nitrergic IJP in global GCKO. In conclusion, our results prove the obligatory role of NO-GC in ICC for the initiation of an IJP. NO-GC in SMC appears to enhance the nitrergic IJP resulting in a stronger and a prolonged hyperpolarization in fundus and colon SMC, respectively. Thus, NO-GC in both cell types is mandatory to induce a full nitrergic IJP. Our data from colon clearly reveal the nitrergic IJP to be biphasic resulting from individual inputs of ICC and SMC.