CMV infection is a major complication after allogeneic HSCT. Although peptide/HLA tetramer and IFN-g ELISPOT assays have been used to measure CMV-specific T cells to monitor the reactivation of CMV, detailed evaluations of these assays and reconstitution kinetics of anti-CMV T cells in RIST and conventional HSCT have not yet been made. In this study, we prospectively monitored CMV-specific T cells after allogeneic peripheral blood HSCT using HLA-tetramer and IFN-g ELISPOT assays specific for previously identified HLA-A*0201 binding pp65 495–503 (NLVPMVATV) peptide in 28 HLA-A*0201- and 9 HLA-A*0206-positive patients. This peptide was able to induce CTL from PBMC in 7 of 8 and 6 of 6 HLA-A*0201- and HLA-A*0206-positive healhy individuals, respectively, indicating that this is an immunodominant CMV peptide for individuals with these HLA-A2 subtypes. The mean of the first day of detectable CMV-specific T cells in 23 patients after allogeneic HSCT was 52.9 days by the HLA-tetramer assay and 29.5 days by the IFN-g ELISPOT assay, indicating that the IFN-g ELISPOT assay is more sensitive than the HLA-tetramer assay, and reconstitution of CMV-specific T cells was detected significantly earlier by the IFN-g ELISPOT assay (p<0.001). The absolute numbers of CMV-specific T cells calculated based on the HLA-tetramer assay were always higher than those with the IFN-g ELISPOT assay, suggesting that the IFN-g ELISPOT assay was able to measure functional T cells. We then compared the recovery of functional CMV-specific T cells using the IFN-g ELISPOT assay among patients who received various HSCT protocols, including RIST without anti-thymocyte globulin (ATG), RIST with ATG, and conventional HSCT. The mean of the first day of detectable functional CMV-specific T cells was 23 days, 39 days, and 79 days, respectively. Thus, the recovery of functional CMV-specific T cells in patients with conventional HSCT was profoundly delayed compared to those in patients who received RIST without ATG (p=0.0001) and RIST with ATG (p=0.02). The recovery of functional CMV-specific T cells in patients who received RIST with ATG was significantly delayed compared to that in patients who received RIST without ATG (p=0.006). We found that patients who had more than 1x10[sup6] functional CMV-specific T cells/L peripheral blood did not develop CMV antigenemia, indicating that this level of recovery was clinically sufficient for protection against CMV infection. The number of functional CMV-specific T cells in patients with RIST without ATG reached 1x10[sup6] cells/L significantly earlier than that in patients who received RIST with ATG (P=0.014). Therefore, RIST without ATG offers the advantage of early reconstitution of anti-CMV immunity. These results indicate that the measurement of functional T cells specific for the single immunodominant CMV peptide using the ELISPOT assay may be useful for evaluating immune recovery for CMV.