Conventional assays for quantification of allo-reactive T-cell precursor frequencies (PF) are relatively insensitive. We present a robust assay for quantification of PF of T-cells with direct donor-specificity, and establish the kinetics of circulating donor-specific T cells after liver transplantation (LTx). B cells from donor splenocytes were differentiated into professional antigen-presenting cells by CD40-engagement (CD40-B cells). CFSE-labelled PBMC from LTx-recipients obtained before and at several time points after LTx, were stimulated with donor-derived or 3rd party CD40-B cells. PF of donor-specific T cells were calculated from CFSE-dilution patterns, and intracellular IFN-γ was determined after re-stimulation with CD40-B cells. Compared to splenocytes, stimulations with CD40-B cells resulted in 3 to 5-fold higher responding T-cell PF. Memory and naïve T-cell subsets responded equally to allogeneic CD40-B cell stimulation. Donor-specific CD4+ and CD8+ T-cell PF ranged from 0.5 to 19% (median: 5.2%). One week after LTx, PF of circulating donor-specific CD4+ and CD8+ T cells increased significantly, while only a minor increase in numbers of T cells reacting to 3rd party allo-antigens was observed. One year after LTx numbers of CD4+ and CD8+ T cells reacting to donor antigens, as well as those reacting to 3rd party allo-antigens, were slightly lower compared to pre-transplant values. Moreover, CD4+ and CD8+ T cells responding to donor-derived, as well as those reacting to 3rd party CD40-B cells, produced less IFN-γ. In conclusion, our alternative approach enables detection of allo-reactive human T cells at high frequencies, and after application we conclude that donor-specific T-cell PF increase immediately after LTx. However, no evidence for a specific loss of circulating T-cells recognizing donor allo-antigens via the direct pathway up to 1 year after LTx was obtained, underscoring the relative insensitiveness of previous assays.