The nontoxic B subunit of cholera toxin (CTB) has been used as an adjuvant in experimental systems of mucosal vaccination. However, the mechanisms behind its adjuvant effects remain unclear. Here, we have used an ex vivo system to elucidate these mechanisms in antigen-specific T cells. Using splenocytes from keyhole limpet haemocyanin (KLH)-immunized mice, initial experiments showed that recombinant CTB (rCTB) did not affect the KLH-specific proliferation of splenocytes isolated from mice immunized 2 weeks earlier. However, rCTB strongly enhanced the KLH-specific proliferation of splenocytes from mice immunized with KLH 4 weeks prior. This adjuvant effect was dose-dependent, with maximum at 30-300 ng/ml rCTB. At higher doses of CTB this effect declined because of the induction of apoptosis. Using antibody depletion and coculture systems, we show that rCTB directly costimulates KLH-specific CD4+ and CD8+ T-cell proliferation but not B cells. Enzyme-linked immunospot (ELISPOT) assays revealed that rCTB also enhanced the KLH-specific CD4+ T-cell-mediated production of interleukin-2 (IL-2), IL-4 and interferon-gamma(IFN-gamma) by four to fivefold. Characterizing the adjuvant effect of rCTB in vivo confirmed the results above, i.e. rCTB mediated a twofold increase in the ex vivo T-cell response when used as a classical adjuvant in a secondary, but not in a primary KLH-immunization regimen. Together these data show that rCTB can act as a strong adjuvant, by directly costimulating antigen-primed CD4+ and CD8+ T cell in a dose-dependent manner. This new insight might be valuable in the future rational design of bacterial toxin-based vaccines.