We have used the potent mucosal immunogen cholera toxin (CT) to assess antigen-specific CD4+ T-cell responses, including Th1- and Th2-type cells in mucosa-associated tissues, e.g. Peyer's patches (PP), and systemic tissue, e.g. spleen (SP), for their regulatory role in the induction of CT-specific B-cell antibody responses in the gastrointestinal (GI) tract as well as in systemic sites. The CT was given by either oral or intravenous (i.v.) routes and the mice orally immunized with CT exhibited brisk IgA anti-CT antibody responses in faecal extracts and elevated IgG anti-CT antibody responses in serum. Further, significant IgA anti-CT spot-forming cells (SFCs) were seen in lamina propria lymphocytes (LPLs) from mice orally immunized with CT. In contrast, i.v. immunization with CT induced IgM and IgG anti-CT SFC responses in SP, and serum anti-CT antibodies of these two isotypes; no anti-CT responses were induced in the GI tract after immunization by this route. The CD4+ T cells isolated from PP and SP of mice orally immunized with CT were stimulated in vitro with CT-B-coated latex microspheres for 1-6 days, and the induction of IL-2 and interferon gamma (IFN-γ) (Th1-type) or IL-4 and IL-5 (Th2-type) producing SFCs were analysed by a cytokine-specific ELISPOT and cytokine-specific mRNA was detected by reverse transcriptase (RT)-PCR assays. Cultures of PP and SP CD4+ cells from orally immunized mice contained elevated numbers of IL-4- and IL-5-SFCs, significant levels of IL-4 and IL-5 mRNA, only small numbers of IFN-γ- and IL-2-SFCs and low to undetectable levels of IFN-γ and IL-2 mRNA. On the other hand, SP CD4+ Th-cell cultures from mice immunized i.v. with CT included both IL-4- and IL-5- as well as IFN-γ- and IL-2-SFCs; no CT-specific Th cells were detectable in PP. These results show that oral immunization with CT preferentially induces CD4+ T cells which produce IL-4 and IL-5 in both PP and SP. Furthermore, the presence of elevated numbers of these Th2-type cells directly correlated with significant IgA anti-CT-B antibody responses in LPLs isolated from the GI tract. © 1994.