We have previously shown that human endothelial cells (EC) are less efficient than professional APC, eg, B lymphoblastoid cells (BLC), at stimulating allogeneic CD8+ T cells to develop into CTL. In this study we describe FACS-based limiting dilution analyses using the dilution of the intracellular dye CFSE as an indicator of CD8+ T cell alloactivation and expansion with significantly increased sensitivity compared with conventional, cytotoxicity-based assays. In addition, this assay permits the relative size of clonal CTL populations that are generated in individual CD8+ T cell cultures to be determined (clonal burst size). We have applied this method to quantitatively compare the generation of CTL at the clonal level following stimulation of allogeneic CD8+ T cells by either BLC or HUVEC derived from the same donor. CD8+ T cells expanded by allostimulation were identified as CD8+, CFSE low cells and were categorized as CTL by the expression of intracellular perforin and IFN-γ. Precursor frequencies for EC-stimulated CTL were 5-to 40-fold (mean, 7.5-fold) lower compared with BLC-stimulated CTL (p< 0.01). Concomitantly, the average clonal burst sizes in EC-stimulated CTL cultures were significantly smaller than those in conventional CTL cultures, primarily due to the occurrence of some very large clone sizes exclusively with BLC stimulation. Although EC-stimulated CTL were generated only from the memory subset of CD8+ T cells, BLC-stimulated very large burst sizes of CTL were observed from both naive and memory CD8+ T cell precursors. These data establish that both a lower frequency of reactive precursors and more limited clonal expansion, but not regulatory T cells, contribute to the reduced capacity of EC to promote alloreactive CTL differentiation compared with that of professional APC.