The antigen specificity of HCMV-specific cytotoxic T cells (CTL) was investigated in healthy adult seropositive donors by analyzing the frequency of CTL precursors (CTL(p)) against six different HCMV-encoded proteins. HCMV-specific, CD8+ CTL clones from two donors were also characterized. The precursor frequency analysis showed that a major phosphoprotein of HCMV, pp65 (UL83), was the immunodominant CTL target antigen in healthy seropositive adults. The frequency of CTL(p) specific for different HCMV proteins ranged from 1/12,000 for pp65 to >1/100,000 for gB (UL55), gH (UL75), pp50 (UL44), and pp28 (UL98). Characterization of the HCMV-specific CTL clones from two donors revealed that the majority or clones lysed targets expressing pp65 and that none of the clones recognized other HCMV proteins.