In vivo replication of rotaviruses is generally limited to enterocytes. Because of this restriction, most blood circulating rotavirus-specific B cells are hypothesized to originate in Peyer's patches and should express the intestinal homing receptor α4β7. To test this hypothesis in humans, we used a flow cytometry assay that identifies antigen-activated (IgD−) B cells (CD19⁺) that express surface rotavirus-specific immunoglobulin. With this assay we could detect rotavirus-specific B cells in both children and adults with an acute rotavirus (RV) infection. Staining with an anti-α4β7 monoclonal antibody, we could determine that B cells that express rotavirus-specific surface immunoglobulin predominantly express α4β7. The response of rotavirus-specific antibody-secreting cells in the peripheral blood of children and adults with acute rotavirus infection was also studied by ELISPOT. The antibody-secreting cells of children were mainly of the IgM isotype, while the antibody-secreting cells of adults were predominantly of the IgA and IgG isotype. α4β7+ and α4β7− subsets of peripheral blood mononuclear cells were purified using paramagnetic beads and then tested in the ELISPOT assay. Rotavirus-specific antibody-secreting cells were predominantly present in the α4β7+ subpopulation. The flow cytometry assay we have described will permit future studies to characterize the phenotype of virus-specific B cells and could be useful in the study of the immunogenicity and protective efficacy of RV vaccines and the identification of markers of protective immunity.