The function of cytotoxic T lymphocytes (CTLs) in rotavirus (RV) infection in humans is poorly understood. To date, no RV-specific human leukocyte antigen (HLA) class I-restricted T-cell epitopes have been described. In this study, four peptides derived from human RV Wa strain VP6 protein were predicted by computer algorithms and verified by an HLA*0201-binding assay. Two peptides with high affinity for HLA-A*0201 molecules were further assessed. The CTLs induced in vitro by P340-348 (TLLANVTAV)-loaded autologous dendritic cells from peripheral blood lymphocytes of HLA-A*0201-matched healthy donors released gamma interferon specifically upon stimulation with P340-348-loaded T2 cells. The CTLs lysed both P340-348-loaded T2 cells and human RV Wa strain-infected HLA-A*0201(+) Caco-2 cells in an antigen-specific and HLA-A*0201-restricted manner. At the same time, P340-348 was shown to be immunogenic in vivo in HLA-A*0201/Kb transgenic mice. It is proposed that P340-348 is an HLA-A*0201-restricted CTL epitope.