The mechanisms which regulate mucosal IgA responses to orally administered protein vaccines are not yet fully elucidated. We have used two delivery systems, soluble tetanus toxoid (TT) with the mucosal adjuvant cholera toxin (CT) and recombinant Salmonella expressing Tox C, a fragment of TT, to assess the nature of CD4+ T helper (Th) cells and derived cytokines which support mucosal IgA responses in both normal and cytokine knockout (interferon gamma knockout; IFN-gamma-/- and IL-4-/-) mice. Our results provide important new information regarding Th cell and cytokine regulation of mucosal IgA responses. Whereas TT coadministered with CT induces predominant TT-specific Th2-type responses, rSalmonella delivery of Tox C induced dominant Th1-type responses along with synthesis of the Th2-cytokine IL-10. Both vaccine regimen elicited high levels of mucosal S-IgA and IL-6 production by macrophages. Further oral immunization of IFN-gamma-/- and IL-4-/- mice with rSalmonella Tox C also induced macrophage-derived IL-6 and Th2-derived IL-10 as well as S-IgA responses, suggesting that IFN-gamma from Th1-type cells as well as traditional Th2 cells producing IL-4 and IL-5 are not essential for mucosal IgA responses. Rather, induction of second level Th2 cells producing IL-10 together with high levels of IL-6 from other cell sources may be sufficient for mucosal IgA responses in the absence of traditional Th2 cells. These studies were facilitated by the development of a sensitive new luminometry assay which allowed detection of cytokines and cell surface molecules which are below the levels of detection by current solid phase assays.