Background Myogenic tone in the lower esophageal sphincter (LES) prevents stomach acid refluxing into the esophagus body1. LES tone has been proposed to be due to activation of Ca2+-activated-Cl- channels (CaCC) leading to the opening of voltage-dependent Cav1.2 Ca2+ channels in LES smooth muscle cells (SMCs). LES-SMC are relatively depolarized, facilitating activation of Cav1.2 channels to sustain contractile tone2.  In other regions of the gut, intramuscular interstitial cells of Cajal (ICC-IM) influence SMC by the activation of CaCC encoded by Ano1. In colon ICC-IM, Ca2+ transients activate Ano1 channels and contribute to setting the resting membrane potential of colonic muscles3,4. Thus, Ca2+ signalling is fundamental for ICC-IM function, yet little to nothing is currently known about Ca2+ signalling in LES ICC-IM or if they express Ano1 channels. We hypothesized that ICC-IM exhibit intracellular Ca2+ signalling that activates Ano1 channels, this then depolarizes electrically connected SMC, to set favorable conditions for activation of Cav1.2 channels and contributes to myogenic tone of the LES.   Methods Immunohistochemistry and isometric tension recordings were performed on strips of mouse and Cynomolgus monkey LES. LES SMCs and ICC-IM were isolated from mice expressing ICC and SMC fluorescent reporters (Kit+/copGFP and SmHC+ /eGFP mice respectively). Ca2+ transients in LES ICC-IM were visualised in-situ in mice expressing GCaMP6f using Cre-LoxP, driven by a Kit promoter). Spatio-temporal map analysis was used to quantify Ca2+ transients5.   Results Immunohistochemistry revealed that Ano1 channels are highly expressed in ICC-IM (Kit+ cells) of the mouse (n=6) and monkey LES (n=8). qPCR data from enriched populations of LES ICC-IM and SMC from fluorescent reporter mice showed expression of enriched Ano1 transcripts occurred almost exclusively in ICC-IM (n=17). ICC-IM imaged from LES tissues of Kit-GCaMP6f mice exhibited stochastic intracellular Ca2+ signaling in situ, firing hundreds of Ca2+ transients min-1 from multiple intracellular sites. Ca2+ transients were abolished by the SERCA pump inhibitor cyclopiazonic acid (P005, n=8). Ca2+ influx played a major role in sustaining Ca2+ transients, as they were reduced after 6 mins in Ca2+ free medium (P0.05) but were reduced in frequency by an Orai channel antagonist (GSK 7975A, n=10, P<0.01). Finally, mouse (n=11) and monkey (n=10) LES strips developed spontaneous tone that was reduced by ~ 60 % (P<0.001) by the Ano1 channel antagonist Ani9.   Conclusions Ano1 channels, expressed in ICC-IM, are activated by dynamic Ca2+ signalling in ICC-IM, contributing to spontaneous tone in the mouse and monkey LES.