Despite the mucosal immunogenicity and adjuvanticity in vivo of cholera toxin (CT), both CT and CT B subunit are strong inhibitors of T cell activation in vitro. This study asked whether such T cell inhibition is relevant to the mucosal effects of CT in vivo. The activation of T cells pulsed in vitro for only 15 to 120 min with CT or CT B subunit, respectively, was inhibited, consistent with the expected short exposure times in vivo. Although both CD8+ and CD4+ T cells were inhibited in vitro, CD8+ T cells bound more toxin and were inhibited to a greater degree than were CD4+ T cells. Intestinal gavage of mice with 10 μg CT did not alter the overall composition of Peyer's Patch, mesenteric lymph node, or spleen but did cause a marked depletion of intraepithelial lymphocytes, mainly CD8+ T cells, and of lymphocytes in the dome epithelium over Peyer's Patch. To determine whether such inhibition oft cells was functionally relevant in vivo, T cells from mice fed keyhole limpet hemocyanin (KLH) were adoptively transferred into naive recipients, who were then parenterally immunized. T cells from mice fed KLH alone inhibited both the systemic IgG and secretory IgA anti- KLH response, but T cells from mice fed KLH plus CT did not, indicating that mucosally applied CT was able to abrogate the induction of this suppressor T cell. We conclude that one of the mechanisms of CT's mucosal effects in vivo is the inhibition of certain mucosal T cell functions and alteration of the regulatory T cell environment in gut-associated lymphoid tissue.