We investigated immune reconstitution against human cytomegalovirus (HCMV) in 57 hematopoietic stem cell transplant (HSCT) recipients, aged 1 to 24 years, through a novel method combining T-cell stimulation by HCMV-infected autologous dendritic cells with simultaneous cytometric quantification of HCMV-specific, IFNgamma-producing CD4(+) and CD8(+) T cells. Lymphoproliferative response (LPR) to HCMV antigens was also determined. Patients were stratified into 2 groups according to HCMV serostatus, comprising 39 HCMV-seropositive (R(+)) and 18 HCMV-seronegative (R(-)) patients who received a transplant from a sero-positive donor. Recovery of both HCMV-specific CD4(+) and CD8(+) T-cell immunity occurred in all 39 R(+) patients within 6 months and in 6 (33%) of 18 R(-) patients within 12 months. In R(+) patients, the median numbers of HCMV-specific CD8(+) and CD4(+)T cells were significantly higher than those of healthy controls, starting from days +60 and +180, respectively. In (R-) patients, the median numbers of HCMV-specific T cells were consistently lower than in R(+) patients. LPR was delayed compared with reconstitution of IFNgamma-producing T cells. Patients with delayed specific immune reconstitution experienced recurrent episodes of HCMV infection. HCMV seropositivity of young HSCT recipients is the major factor responsible for HCMV-specific immune reconstitution, irrespective of donor serostatus, and measurement of HCMV-specific T cells appears useful for correct management of HCMV infection.