We used normal, CD4 and CD8 gene-targeted mice to investigate the role of CD4+ and CD8+ T cells in the regulation of gut mucosal immune responses following oral immunizations with cholera toxin (CT) adjuvant. Phenotypic analysis of mucosa-associated tissues revealed normal CD3+ T-cell frequencies in CD4−/− and CD8−/− mice such that in CD4−/− mice the CD8+ and double-negative (DN) T cells were increased. In CD8−/− mice the CD4+ T cells were increased, with the exception that in the intraepithelial compartment the CD3+ T cells were predominantly DN γδ T-cell receptor (TCR)+ T cells. All mice, normal and deficient, failed to respond to oral immunization with the antigen, keyhole limpet haemocyanin (KLH), alone. In the presence of CT adjuvant, however, CD8−/− mice consistently exhibited three- to fivefold stronger gut mucosal responses compared to normal C57Bl/6 mice. By contrast, no difference was observed for systemic responses between CD8−/− and normal mice. Thus the up-regulation selectively affected mucosal responses, suggesting that, contrary to the CD8−/− mouse gut, the normal gut mucosa may host CD8+ T cells that exert a local suppressive effect on T- and B-cell responses. The magnitude of the enhancing effect of CT was comparable in CD8−/− and normal mice, clearly demonstrating that the adjuvant mechanism of CT does not require CD8+ T cells. On the other hand, the adjuvant effect of CT required CD4+ T cells, because no or poor anti-KLH responses were observed in CD4−/− mice. In both normal and CD8−/− mice CT adjuvant promoted KLH-specific CD4+ T-cell priming without any selective effect on the differentiation towards a T-helper type-1 (Th1) or Th2 dominance. Furthermore, CT adjuvant increased the frequency of CD4+ T cells expressing a memory phenotype, i.e. CD44high, LECAM-1low and CD45RBlow. We have shown, using gene-targeted mice, that CD8+ T cells are not required for the adjuvant effect of CT, and that CD8+ T cells may exert local mucosal down-regulation of intestinal immune responses.