Human cytomegalovirus (HCMV) is one of the most common causes of congenital infection leading to birth defects, and a leading cause of serious illness in patients with impaired cell-mediated immunity. Helper T cell (T(h)) responses to HCMV proteins are likely to be important in limiting viral replication and preventing disease. Previous studies from this laboratory have demonstrated that the amino-terminal 513 amino acids of HCMV glycoprotein B (gB) can stimulate both B and T cell responses in humans. In the present study, the proliferative responses of HCMV-specific T(h) clones to recombinant proteins and synthetic peptides were examined to identify four T(h) epitopes on gp93, which represents the amino-terminal 460 amino acids of the gB polypeptide. Using clones of known HLA restriction specificity from several donors, it was shown that each HLA class II allele preferentially associates with a different epitope on gB. Five clones from two different donors recognized an epitope in the region of amino acids 250 to 264 restricted by DR4Dw14, two clones from different donors recognized an epitope in the region of amino acids 420 to 434 restricted by DR7Dw17, two clones from different donors recognized an epitope in the region of amino acids 178 to 194 restricted by DQw1 and a single clone recognized an epitope in the region of amino acids 190 to 204 restricted by DPw4. Although all peripheral blood mononuclear cells (PBMCs) expressing a particular HLA class II allele were able to present the appropriate HLA-restricted gB peptide to gB-specific T(h) clones, not all individuals expressing a given HLA allele exhibited PBMC responses to the corresponding gB peptide. The HLA-related differences in T(h) recognition of specific epitopes on gB described in this report may have important implications in virus-host interactions and vaccine strategies.