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The pathogenesis of colorectal neoplasia (CRN) has been associated with altered non-neuronal acetylcholine (ACh) metabolism. The aim of this study was to characterize expression, function and cellular location of ACh-related proteins in biopsies obtained from endoscopic normal appearing sigmoid colon in patients with and without CRN. Messenger-RNA (mRNA) levels of 17 ACh-related proteins were quantified by rt-qPCR. Functional responses to ACh, measured as electrogenic transepithelial short circuit current (SCC), were recorded using the Ussing chamber technique. Finally, cellular localization of choline transporter-like proteins (CTLs) and butyryl-cholinesterase enzyme (BChE) was determined by immunohistochemistry. mRNA expression of CTL1 and CTL4 was increased in patients with CRN (p = 0.002 and p = 0.04, respectively). In functional experiments, baseline SCC was increased in CRN patients. ACh induced rapid biphasic changes in SCC. An initial decreasing phase was observed in the minority of CRN patients vs. the majority of controls (25% vs. 69%, respectively, p = 0.031). For the second increasing phase of SCC, data indicated ACh-activation of two receptors. For both parts of the biphasic response, the half maximal effective concentration and maximal responses showed no difference between patient groups. Immunohistochemistry demonstrated CTL1, 3 and 4 and BChE to be localized to colonic crypt cells. We conclude that CRN is associated with increased expression of CTL1 and CTL4, augmented basal prostaglandin-dependent secretion, and altered functional channel response to ACh in human endoscopic normal appearing colonic mucosa. The immunohistochemical findings support CTL1, CTL3, CTL4 and BChE to be involved in non-neuronal mucosal ACh metabolism. This article is protected by copyright. All rights reserved