Human cytomegalovirus (HCMV) is associated with significant morbidity and mortality following immunosuppression and in pregnancy. HCMV infection may be accompanied by acute disease but persists asymptomatically. Cytotoxic T lymphocytes (CTL) appear to be an important immune effector mechanism in maintaining the normal host-virus equilibrium. Glycoprotein B may be an important target for future subunit vaccines as it has been found to elicit both neutralising antibody and CTL responses. We therefore studied the ability of normal asymptomatic HCMV-seropositive individuals and women throughout pregnancy to determine the presence of HCMV and gB-specific CTL responses. CTL effector cells were induced by stimulation of peripheral blood mononuclear cells (PBMC) with AD169 HCMV-infected cells and gB-specific CTL were identified using chromium labeled, vac.gB-infected cells. In 7 HCMV-seropositive individuals, HCMV-specific CTL were identified. Three of the 7 individuals which lysed HCMV-infected cells lysed vac.gB-infected B cells. However, vac.gB-infected autologous fibroblasts, which only present MHC class I, were not killed. Using MHC class I single allele targets, no specific lytic response was observed, suggesting a MHC class II restricted CTL response. Flow cytometric analysis showed the gB-specific effector cell phenotype to be CD3+, CD4+, CD8-. In conclusion, a gB-specific CTL lytic response was identified in seropositive individuals which in most cases was MHC class II-restricted.