The release of Bacillus anthracis spores can cause significant morbidity and mortality and an effective vaccine as well as directed immunotherapeutics are needed. The currently licensed vaccine requires six primary vaccinations, annual boosters, and has been associated with serious adverse events. Thus there is increasing interest in developing an improved vaccination strategy which can generate lasting protective immunity with reduced vaccinations. This study seeks to identify the major humoral targets of B. anthracis that provide protection. To this end, plasma was obtained from two hundred vaccinated individuals and thirty unvaccinated controls. Ninety-five percent of all vaccinated individuals had anti-protective antigen antibodies and these antibodies correlated with in vitro protection against lethal toxin. Utilizing overlapping decapeptides, we identified common antigenic regions within those individuals with high toxin neutralizing activity. Epitope-specific antibodies directed against three of these regions were isolated and were able to mediate protection in vitro and in a mouse model of in vivo lethal toxin challenge. Identification of common, protective, peptide-specific humoral immune responses provide key information for further immunotherapeutic and vaccine development. NIAID: 5U19AI062629