The intestinal homing receptor, alpha(4)beta(7), helps target lymphocytes to Peyer's patches (PP) and intestinal lamina propria (ILP). We have previously shown that protective immunity to rotavirus (RV), an intestinal pathogen, resides in memory B cells expressing alpha(4)beta(7). In this study, using a novel FACS assay, we have directly studied the phenotype of B cells that express surface RV-specific Ig during the in vivo RV immune response. During primary infection, RV-specific B cells first appear as large IgD(-)B220(low)alpha(4)beta(7)(-)and alpha(4)beta(7)(+) cells (presumptive extrafollicular, Ab-secreting B cells), and then as large and small IgD(-)B220(high)alpha(4)beta(7)(-)cells (presumptive germinal center B cells). The appearance of B cells with the phenotype of large IgD(-)B220(low)alpha(4)beta(7)(+) cells in PP and most notably in mesenteric lymph nodes coincides with the emergence of RV-specific Ab-secreting cells (ASC) in the ILP. Thus, these B lymphocytes are good candidates for the migratory population giving rise to the RV-specific ASC in the ILP. RV-specific long-term memory B cells preferentially accumulate in PP and express alpha(4)beta(7). Nine months after infection most RV-specific IgA ASC are found in PP and ILP and at lower frequency in bone marrow and spleen. This study is the first to follow changes in tissue-specific homing receptor expression during Ag-specific B cell development in response to a natural host, tissue-specific pathogen. These results show that alpha(4)beta(7) is tightly regulated during the Ag-specific B cell response to RV and is expressed concurrently with the specific migration of memory and effector B cells to intestinal tissues.