Cell-mediated immunity is essential for control of human cytomegalovirus (HCMV) infection. We used a pool of 138 synthetic overlapping pentadecapeptides overspanning the entire pp65 protein to generate polyclonal CMV-specific T-cell lines from 12 CMV-seropositive donors inheriting different HLA genotypes. Autologous monocyte-derived dendritic cells (DCs) pulsed with this complete pool consistently induced highly specific T cells that selectively recognized 1-3 pentadecapeptides identified by secondary responses to a mapping grid of pentadecapeptide subpools with single overlaps. Responses against peptide-loaded targets sharing single HLA class I or II alleles identified the restricting HLAalleles. HLA-A*0201⁺ donors consistently responded to pentadecapeptides containing HLA-A*0201-binding epitope_aa495-503NLVPMVATV. T-cell lines from other donors contained high frequencies of CD4 and/or CD8 T cells selectively reactive against peptides presented by other HLA alleles, including both known epitopes such as _aa341-350QYDPVAALF (HLA-A*2402) as well as unreported epitopes such as _aa267-275HERNGFTVL (HLA-B*4001 and B*4002) and _aa513-523FFWDANDIYRI (HLA-DRB1*1301). These T cells consistently lysed CMV-infected target cells. Thus, this approach fosters expansion and selection of HLA-restricted CMV-pp65–reactive T-cell lines of high specificity that also lyse CMV-infected targets, and from a functional and regulatory perspective, may have advantages for generating virus-specific T cells for adoptive immunotherapy.