We studied neuroendocrine mediator regulation of adhesion and motility of human epidermal keratinocytes (EK) and found that cholinergic compounds control EK cell‐matrix and cell‐cell attachment. In this study, we tested the anti‐acantholytic activity of muscarinic agonists in pemphigus and non‐pemphigus acantholysis, and investigated the effects of pemphigus antibody (Pab) on the the keratinocyte muscarinic acetylcholine receptors (mAChR). Acantholysis produced by Pab from two patients with pemphigus vulgaris was compared with acantholysis induced by the serine protease trypsin. the calcium chelator EOT A or the muscarinic antagonist atropine. Trypsinized EK first lost contact with microplate surface and then retracted their intercellular filaments. EDTA‐treated cells first detached from each other and then from the dish. EK cultures treated with Pab or atropine rounded up and retracted their intercellular filaments simultaneously, although it took hours to obtain acantholysis with Pab treatment compared to several seconds with atropine treatment. Addition of acetylcholine or other muscarinic agonists (bethanechol. carbachol or methacholine) to acantholytic cultures reversed both pemphigus and non‐pemphigus acantholysis. Acantholysis induced by atropine reversed spontaneously. Short‐term preexposure of EK to Pab significantly increased, and long‐term preexposure significantly decreased, the [³H]atropine binding to keratinocyte mAChR. We conclude that muscarinic agonists reverse various types of acantholysis. including acantholysis induced by Pab, and that binding of Pab to EK may affect the ability of keratinocyte mAChR to bind its ligands.