A CTL antigenic site located between residues 273 and 291 of the E1 envelope protein of RV was identified by 51Cr-release assays employing SPs. Two E1-specific CTL clones were examined for immune recognition of RV wild-type and attenuated vaccine strains and recombinant E1 protein. The exact sequence (273-284) recognized by both clones was delineated by using truncated and overlapping SPs covering these residues. The defined T-cell site overlapped almost completely with a virus neutralizing antibody-binding site previously identified with mouse monoclonal and human antibodies. A series of single aa-substituted SP analogues of E1(273-284) was used to define residues critical for T-cell recognition. Using EBV-BL displaying different HLA-DR haplotypes and -DR4 subtypes as targets to determine MHC class II restriction elements, immune recognition by both T-cell clones was shown to be associated with HLA-DR4. Three HLA-DR4 subtypes (DR4Dw13A, DR4Dw13B, and DR4KT2) sharing a common residue, glutamic acid at position 74 in their beta 1 chains, were able to present SP E1(273-284) to the T-cell clones.