Different types of anthrax toxin neutralization assays have been utilized to measure the antibody levels elicited by anthrax vaccines in both nonclinical and clinical studies. In the present study, we sought to determine whether three commonly used toxin neutralization assays-J774A.1 cell-, RAW 264.7 cell-, and CHO cell-based assays-yield comparable estimates of neutralization activities for sera obtained after vaccination with anthrax vaccines composed of recombinant protective antigen (rPA). In order to compare the assays, sera were assayed alongside a common reference serum sample and the neutralization titers were expressed relative to the titer for the reference sample in each assay. Analysis of sera from rabbits immunized with multiple doses of the rPA vaccine showed that for later bleeds, the quantitative agreement between the assays was good; however, for early bleeds, some heterogeneity in relative neutralization estimates was observed. Analysis of serum samples from rabbits, nonhuman primates, and humans immunized with the rPA vaccine showed that the relative neutralization estimates obtained in the different assays agreed to various extents, depending on the species of origin of the sera examined. We identified differences in the magnitudes of the Fc receptor-mediated neutralization associated with the J774A.1 cell- and RAW 264.7 cell-based assays, which may account for some of the species dependence of the assays. The differences in the relative neutralization estimates among the assays were relatively small and were always less than 2.5-fold. However, because toxin neutralization assays will likely be used to establish the efficacies of new anthrax vaccines, our findings should be considered when assay outputs are interpreted.