Adoptive immunotherapy with cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) is an effective strategy for preventing and treating viral reactivation after allogeneic stem cell transplantation (SCT). We have shown previously that CMV CTL can be generated in 1 to 2 weeks by stimulating donor lymphocytes with peptide mixes derived from full-length pp65 and IE1. We conducted a multi-institutional study of CMV-specific CTL for patients with persistent or anti-viral-resistant CMV infections after allogeneic SCT, to determine the safety, feasibility, and immunologic effects of this approach. We were successful in stimulating CTL from 10/10 donors with pooled CMV overlapping peptide mixes. Five of the 7 subjects who met infusion criteria had new onset CMV-specific CTL activity detected within 4 to 6 weeks after infusion. Of the 2 subjects who did not have immunologic responses after infusion, 1 received CTL with a low viability after thawing, and the other patient received cyclosporine A and systemic corticosteroids at the time of the infusion, achieving only a low, transient increase (10%) in pp65-specific activity. There was no graft-versus-host disease attributable to these infusions. These findings indicate that the infusion of CTL stimulated over 1 to 2 weeks with overlapping CMV peptides can result in virus-specific immune reconstitution in SCT recipients, without exacerbations of graft-versus-host disease.