We have shown previously that cultured HUVEC or mixtures of endothelial cells (EC) and B lymphoblastoid cells (BLC) induce the differentiation of purified CD8+ PBL into allospecific, class I MHC-restricted CTL that lyse EC, but not BLC autologous to EC. Furthermore, these EC-selective CTL lines secrete little IFN-γ after target cell contact. In the present study, we have analyzed these polyclonal populations at a single cell level by cloning at limiting dilution and propagating the resulting CTL clones in the absence of EC. Phenotypically stable, alloreactive EC-selective CTL preferentially emerge from cocultures in which EC or EC+ BLC are the initial stimulating cell types compared with cocultures stimulated by BLC alone (p= 0.005). Compared with BLC-stimulated CTL, EC-stimulated CTL clones often fail to secrete IFN-γ after target cell contact (p= 0.0006) and constitutively express CD40 ligand (CD40L) at rest (p= 0.0006). The absence of IFN-γ secretion does not result from a switch to IL-4 secretion. The expression of CD40L inversely correlates with the secretion of IFN-γ after target cell contact (p= 0.0001), but correlations of CD40L expression and failure to secrete IFN-γ with EC-selective killing did not reach statistical significance. We conclude that in a microenvironment in which allogeneic EC are in close contact with infiltrating CD8+ T cells, such as within a graft arterial intima, CTL subsets may emerge that display EC selectivity or express CD40L and secrete little IFN-γ after Ag contact.