Pemphigus vulgaris (PV) is a potentially fatal autoimmune mucocutaneous blistering disease. It was assumed that PV is caused by anti-desmoglein (Dsg) 3 autoimmunity because absorption of PV sera with a chimeric baculoprotein containing the Dsg 3 and IgG1 portions, rDsg3-Ig-His, eliminated disease-causing antibodies. In this study we demonstrate that rDsg3-Ig-His adsorbs out autoantibodies to different keratinocyte antigens, including a non-Dsg 3 130-kd polypeptide. Because the pool of disease-causing PV IgGs contains antibodies against the keratinocyte acetylcholine receptor (AChR), we sought to identify the targeted receptor(s). Preincubation of monkey esophagus with PV antibodies blocked specific staining of the keratinocyte cell membrane with rabbit monoepitopic antibody to α9 AChR, indicating that this first of its kind AChR with dual, muscarinic and nicotinic pharmacology is targeted by PV autoimmunity. Anti-α9 antibody stained keratinocytes in a fishnet-like intercellular pattern, and visualized a single band at ∼50 kd in Western blots of keratinocyte membrane proteins. Using step-by-step reverse transcription polymerase chain reactions with primers based on known α9 sequence regions, we identified the complete reading frame of human α9. Its amino acid sequence showed 85% similarity with rat α9. Treatment of keratinocyte monolayers with anti-α9 antibody induced pemphigus-like acantholysis, which could be reversed either spontaneously or by using the cholinergic agonist carbachol. We conclude that α9 is coupled to physiological regulation of keratinocyte adhesion, and its interaction with PV IgG may lead to blister development.