Donor-induced and third-party-induced proliferation of T-helper and T-cytotoxic (Tc) cells and their naïve and memory subsets was evaluated simultaneously in single blood samples from 77 children who received steroid-free liver transplantation (LTx) after induction with rabbit anti-human thymocyte globulin. Proliferation was measured by dilution of the intravital dye carboxyfluorescein succinimidyl ester (CFSE) in a 3- to 4-day mixed lymphocyte response coculture. The ratio of donor/third-party-induced proliferated (CFSE(low)) T-cells was reported as the immunoreactivity index (IR) for each subset. Rejectors were defined as those who experienced biopsy-proven acute cellular rejection within 60 days of the assay. IR > 1 signified increased risk of rejection, and IR < 1 implied decreased risk. Demographics for 32 rejectors and 45 nonrejectors were similar. Proliferated CFSE(low) T-cells and subsets were significantly higher among rejectors compared with nonrejectors. In 33 of 77 randomly selected children, logistic regression, leave-one-out cross-validation, and receiver operating characteristic analyses showed that the IR of Tc cells was best associated with biopsy-proven rejection (sensitivity > 75%, specificity > 88%). Sensitivity and specificity were replicated in the remaining 44 children who composed the validation cohort. IR of CFSE(low) Tc cells correlated significantly with IR of proinflammatory, allospecific CD154+ Tc cells (r = 0.664, P = 0.0005) and inversely with IR of allospecific, anti-inflammatory, cytotoxic T lymphocyte antigen 4-positive Tc cells (r = -0.630, P = 0.007). In conclusion, proliferative alloresponses of Tc cells can identify rejection-prone children receiving LTx. Liver Transpl 15:978-985, 2009. (c) 2009 AASLD.