To address the controversy surrounding the role of interstitial cells of Cajal (ICC) in nitrergic neurotransmission to gastrointestinal smooth muscle, circular smooth muscle from the lower esophageal sphincter (LES) of W/Wvwild-type and mutant (ICC-deficient) mice were studied by using intracellular and tension recordings in vitro. Resting membrane potential was more negative, and the spontaneous unitary potentials diminished in mutant mice. In wild-type mice, nerve stimulation induced a biphasic inhibitory junction potential (IJP) consisting of a fast initial IJP followed by a long-lasting slow IJP (LSIJP). The IJP was markedly impaired in a significant proportion of mutant mice, whereas in others it was normal. Pharmacological studies in the mice with markedly impaired IJPs revealed that cholinergic and purinergic components of the nerve-mediated responses appeared intact. In wild-type mice, caffeine hyperpolarized smooth muscle cells, inhibited the initial fast IJP, and completely abolished the LSIJP. In mutant mice, caffeine depolarized smooth muscle cells and abolished the impaired LSIJP but did not affect the initial fast IJP. Immunohistochemical staining for c-Kit confirmed deficiency of ICC in mutant mice with a normal nitrergic IJP. Rings of LES circular smooth muscle from W/Wvmutant mice generated significantly less spontaneous tone than controls. When tone was restored with carbachol, normal nitrergic LES relaxation was recorded. These data suggest that 1) there is significant variability in the generation of nitrergic neurotransmission in the LES of W/Wvmutant mice, whereas purinergic and cholinergic neurotransmission are intact; 2) the altered nitrergic responses appear to be associated with abnormal Ca2+-dependent signaling initiated by spontaneous Ca2+release from sarcoplasmic reticulum in smooth muscle cells; and 3) c-Kit-positive ICC are not essential for nitrergic neurotransmission in mouse LES smooth muscle.