This study describes a four-color flow cytometric assay that detects CD4+ T cell responses to the anthrax vaccine. Whole blood from seven volunteers who previously obtained the anthrax vaccine was inoculated in vitro with varying concentrations of the anthrax antigen. TNF-alpha and IFN-gamma production from memory CD4+ T cells were measured and compared to a control group who never received the anthrax vaccine.. The optimal antigen concentration for TNF-alpha was determined to be around 7.5 mug/ml and IFN-gamma production was not detected. This assay will be used in future larger prospective studies to further evaluate the cellular immune responses induced by the anthrax vaccine. (C) 2003 Elsevier B.V. All rights reserved.