Epstein-Barr virus (EBV) DNA levels in whole-blood samples of 54 pediatric patients receiving T-cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT) in 2003 to 2007 were retrospectively compared with EBV DNA loads in peripheral blood mononuclear cells (PBMC). Determination of EBV DNA in whole blood missed 1 of 19 patients (5.2%), who tested positive for EBV DNA in PBMC. The analytical sensitivity of EBV DNA detection in whole-blood samples relative to that in PBMC was 94.7%. Regression analysis showed a significant correlation between DNA levels in PBMC and whole blood (r = 0.81; P < 0.001). Relative to that in PBMC, the appearance of EBV DNA in whole blood was delayed in 9/18 patients (median, 49 days; range, 6 to 226 days), while peak levels and clearance were reached simultaneously. Following peak levels, EBV DNA showed a slower decline in whole blood than in PBMC. In conclusion, (i) EBV DNA levels in PBMC were significantly correlated with those in whole blood; (ii) a differential kinetics of EBV DNA in the two blood compartments was observed; and (iii) monitoring of EBV DNA levels in whole blood appears to be a valuable alternative to PBMC in the follow-up of pediatric recipients of haploidentical T-cell-depleted HSCT.