Paraneoplastic pemphigus (PNP) shows autoantibodies mainly to plakin and desmosomal cadherin family proteins. We have recently identified alpha-2-macroglobulin-like-1 (A2ML1), a broad range protease inhibitor, as a unique PNP antigen. In this study, we tested a large number of PNP sera by various methods. Forty (69.0%) of 58 PNP sera recognized A2ML1 recombinant protein expressed in COS7 cells by immunofluorescence and/or immunoprecipitation/immunoblotting. Immunoprecipitation/immunoblotting showed higher sensitivity than immunofluorescence. In addition, 22 (37.9%) PNP sera reacted with A2ML1 by immunoblotting of cultured normal human keratinocytes (NHKs) under non-reducing condition. Statistical analyses using various clinical and immunological data showed that the presence of anti-A2ML1 autoantibodies was associated with early disease onset and absence of ocular lesions. Next, to investigate the pathogenic role of anti-A2ML1 antibody, we performed additional functional studies. Addition of anti-A2ML1 polyclonal antibody to culture media decreased NHK cell adhesion examined by dissociation assay, and increased plasmin activity detected by casein zymography, suggesting that anti-A2ML1 antibody may decrease NHK cell adhesion through plasmin activation by inhibition of A2ML1. This study demonstrates that autoantibodies to A2ML1 are frequently and specifically detected and may play a pathogenic role in PNP.Journal of Investigative Dermatology accepted article preview online, 13 February 2013; doi:10.1038/jid.2013.65.