The human large intestinal epithelium comprises millions of invaginations called colonic crypts, which are highly innervated by cholinergic neurons. Spatiotemporal characteristics of cholinergic-induced calcium waves along the human colonic crypt-axis suggest that calcium signals play a central role in co-ordinating the physiological function and perpetual renewal of the human colonic epithelium. However, the mechanism of colonic crypt calcium signalling is unknown. The aim of the current study was to characterise the cellular and molecular basis of human colonic crypt calcium signal generation. Methods: Human colonic crypts were isolated from colorectal biopsy samples (NRES approval) and placed in a 3D culture system. Intracellular calcium was monitored by Fura2 ratio imaging. mRNA expression was assessed by real-time RT-PCR and protein localisation was visualised by fluorescence immunolabelling and confocal microscopy. Results: Application of carbachol (10 μM) initiated calcium signals at the apical pole of M3AChR-positive cells located in the stem cell niche at the colonic crypt-base. Calcium signals spread from the apical to basal pole of initiator cells and from initiator cells to all neighbouring cell types (e.g. goblet cells) along the crypt-axis. The absence of extracellular calcium attenuated the duration of the biphasic calcium response at the crypt-base, but did not alter the initial peak amplitude (n=5). Thapsigargin pre-treatment did not abolish the carbachol-induced calcium signal at the apical pole of crypt cells (n=14). Immunolabelling revealed the presence of ER (anti-Kdel) at the basal pole and lysosomes (anti-LAMP1) at the apical pole of polarised crypt cells (n=16). Lysosomal lysis by GPN (200 μM) invoked a calcium increase at the apical pole and abolished subsequent responses to carbachol stimulation (n=5), as did pre-treatment with other lysosomotropic agents, chloroquine (100 μM, n=4) and bafilomycin (5 μM, n=4). Colonic crypts expressed mRNA for InsP3R1-3, RyR1&2 and TPC1&2 intracellular calcium channels (N=5 subjects). Carbachol-induced calcium signals were profoundly inhibited (peak amplitude inhibition >80%, P<0.05) by the PLC inhibitor U73122 (10 μM, n=15) or the NAADP antagonist NED-19 (20-200 μM, n=10) and partially inhibited (peak amplitude inhibition <40%, P<0.05) by InsP3R (Xestospongin C, n=5;2-APB, n=10) or RyR (ryanodine, n=10; 8-bromo cADP ribose, n=10) antagonists. Conclusions: Cholinergic stimulation of M3AChR mobilises calcium from lysosomes located at the apical pole of cells within the colonic crypt stem cell niche. The effects of pharmacological agents suggest that dynamic interplay between NAADP-, InsP3- and cADP ribose-sensitive stores determines the spatiotemporal characteristics, and the physiological outcomes, of colonic crypt calcium signalling.