Anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. PA is the major component in the current anthrax vaccine, but the antigenic epitopes on it are not well-defined. We generated a pool of toxin-neutralizing anti-PA monoclonal antibodies (MAbs) to analyze the neutralizing epitopes of PA. Nine toxin-neutralizing MAbs obtained were found bound to three different domains of PA respectively, among which three MAbs with the strongest toxin-neutralizing activity recognized the same epitope within domain 2. This epitope was fine mapped to the chymotrypsin-sensitive site, (SFFD315)-S-312, in the 2 beta(2)-2 beta(3) loop of PA, using phagedisplayed random peptide libraries and mutation analysis. The result demonstrated for the first time that the 2 beta(2)-2 beta(3) loop, which is involved in the transition of PA oligomers from prepore to pore, contains a dominant neutralizing epitope. This work contributes to the immunological and functional analysis of PA and offers perspective for the development of a new epitope vaccine against anthrax. (c) 2006 Elsevier Inc. All rights reserved.