Cholera toxin (CT), the enterotoxin of Vibrio cholerae, is a potent mucosal immunogen as well as a strong mucosal adjuvant to related and unrelated antigens. The mucosal immune response to CT is T cell dependent and MHC class II restricted. The epitopes on CT recognized by T cells have not been identified. The purpose of this study was to determine the fine specificity of T cell recognition of both the CT A subunit (CT-A) and the CT B subunit (CT-B) by using a range of synthetic peptides. After immunization with CT-B or CT-A in CFA subcutaneously, the peripheral lymph node T cells were stimulated with different synthetic peptides in vitro. The peptide specificity of T cell recognition was identified by assaying T cell proliferation and interleukin-3 production. T cells from C57BL/6 (H-2b) high responder mice recognized one immunodominant epitope (peptide 89-100) and one weak epitope (peptide 31-50) on CT-B and two epitopes (peptide 21-39 and 180-194) on CT-A. The immunization of C57BL/6 mice with synthetic immunodominant CT-B peptide 89-100 induced T cell immunity to the pentameric CT-B. Induction of tolerance to CTB peptide 89-100 by i.v. injection in high responder C57BL/6 mice induced unresponsiveness to mucosal immunization with CT, compatible with an immunodominant role for this T cell epitope.