Recovery of cytomegalovirus (CMV)-specific T-cell-mediated immunity after allogeneic hematopoietic stem cell transplantation (SCT) is critical for protection against CMV disease. The study used fluorochrome-conjugated tetrameric complexes of HLA-A2 molecules loaded with the immunodominant NLVPMVATV (NLV) peptide derived from the CMV protein pp65 to quantify A2-NLV-specific CD8+ T cells in partially T-cell-depleted grafts administered to 27 HLA-A*0201+ patients and to monitor recovery of these T cells during the first 12 months after SCT. None of the 9 CMV-seronegative patients became infected with CMV, whereas 14 of 18 CMV-seropositive patients developed CMV antigenemia after SCT. CMV-seropositive recipients of grafts from CMV-seronegative donors required more preemptive treatment with ganciclovir (GCV) than those of grafts from CMV-seropositive donors (3 [1-6] versus 1 [0-3] courses, respectively; P =.009). The number of A2-NLV-specific CD8+ T cells in the grafts correlated inversely with the number of preemptive GCV courses administered (r = -0.61; P =.01). None of the 9 CMV-seronegative patients mounted a CMV-specific immune response as measured by monitoring A2-NLV-specific CD8+ T cells after SCT. Thirteen of 14 CMV-seropositive patients without CMV disease recovered these T cells. In spite of preemptive GCV treatment, CMV disease developed in 4 patients, who all failed to recover A2-NLV-specific CD8+ T cells after SCT (P =.002). Thus, enumeration of HLA-restricted, CMV-specific CD8+ T cells in the grafts and monitoring of these T cells after SCT may constitute a rapid and sensitive tool to identify SCT recipients at risk for developing CMV disease.