Reconstitution of human cytomegalovirus (HCMV) T-cell immunity is crucial in hematopoietic stem cell transplant (HSCT) recipients. The QuantiFERON-CMV assay for cellular HCMV-specific immunity was evaluated in allogeneic HSCT recipients (n = 43) and patients with hematological malignancies (n = 29) attending a tertiary-care Irish hospital. An intracellular cytokine (ICC) assay correlated with the QuantiFERON-CMV assay. Although there was agreement between HCMV seropositivity and QuantiFERON-CMV assay, six HCMV seropositive immunosuppressed patients with hematological malignancy had negative QuantiFERON-CMV results. The 43 HSCT recipients were classified as high risk (D(-)/R(+)) (n = 18), intermediate risk (D(+)/R(+) and D(+)/R(-)) (n = 17), and low risk (D(-)/R(-)) (n = 8). During episodes of HCMV DNAemia no evidence of HCMV-specific immunity was found using the QuantiFERON-CMV assay. Furthermore, the recovery of HCMV-specific CD8(+) T-cell responses in high-risk seropositive recipients of matched unrelated donors was severely delayed, a mean of 200 (SD = 117) days compared to 58 (SD = 23) days for sibling donors (P < or = 0.028). In addition, three patients with late HCMV infection (infection >100 days post-transplant) had delayed reconstitution of HCMV-specific CD8(+) T cells. Interestingly, two recipients (R(+)/D(-)) developed rapid immune reconstitution by days 15 and 36 post-HSCT, suggesting HCMV-specific T-cell lymphopoiesis of recipient origin. Levels of CD8(+) T-cell immunity in HCMV seropositive HSCT recipients were lowest following HSCT. A high number (33%) of indeterminate results was observed immediately after transplantation. Patients with indeterminate QuantiFERON-CMV results had low levels of HCMV-specific CD8(+) T cells. J. Med. Virol. 82:433-440, 2010. (c) 2010 Wiley-Liss, Inc.