Although a T-cell response in human cytomegalovirus (HCMV)-immune individuals exists against the most abundantly expressed protein pp65 of the virus matrix, less is known about the determinants that evoke this response. The aim of the study was to identify regions within HCMV pp65 (ppUL83) that contain sequences for the cellular immune response by the use of three recombinant overlapping beta-galactosidase pp65 fusion proteins (C74, C35, and C47), covering the C-terminal 265 amino acids of the entire pp65 sequence. Two T-cell epitope determinants were recognized by human lymphocytes of healthy, HCMV-seropositive, human leukocyte antigen (HLA)-typed individuals. One T-cell determinant (amino acids [aa] 303-388) was localized in the mid-region of the entire pp65 sequence and a second T-cell determinant (aa 477-561) within the C-terminal region. By fine mapping with synthetic hexadecamer peptides three T-cell epitopes were identified within these two regions: P10-I (aa 361-376) in the mid-region, P3-II (aa 485-499), and P6-II (aa 509-524) in the C-terminal region. Inhibition studies with monoclonal antibodies to HLA class I or class II revealed a class II restricted response to peptides P10-I or P6-II, respectively. P10-I responders shared the HLA-DR11 allele and P6-II responders the -DR3 allele. Therefore, these T-cell epitopes of HCMV pp65 might be presented in association with particular HLA class II alleles.