Serially passaged human endothelial cell (EC) cultures will stimulate highly purified peripheral blood CD4+ T cells to proliferate if and only if the EC cultures are pretreated with IFN-[gamma] to induce de novo expression of MHC class II molecules, principally HLA-DR. HLA-DR-expressing EC alone appear sufficient to stimulate purified CD4+ T cell proliferation without the involvement of other leukocyte populations, as indicated by the following observations:(1) we find no contaminating leukocytes in our EC cultures by FACS analysis or fluorescence microscopy; specifically, there are no detectable CD45 or HLA-DR expressing cells;(2) neither the EC cultures nor the purified CD4+ T cells contain HLA-DR expressing cells detectable by polymerase chain reaction (PCR) of reverse-transcribed mRNA;(3) the stimulatory capacity of the EC cultures is maintained through serial subculture and through low-density replating, indicating that the stimulatory cell type must proliferate in culture as well as EC; and (4) in contrast to MLRs, the response to EC cultures is not inhibited by pretreatment of the stimulator cells and/or responding T cells with the monocyte toxin L-leucine-O-methyl ester. We have used mAb to investigate the role of various EC and T cell surface molecules in the T cell response. mAb to HLA-DR and CD4 inhibit proliferative responses of CD4+ T cells to EC cultures, as would be expected if T cells recognize and proliferate to IFN-[gamma]-nduced allogeneic class II MHC molecules; whereas, also as expected, mAb to class I MHC molecules were without effect. Proliferation is also inhibited by mAbs to T cell CD2 and LFA-1 [beta] chain (CD 18) and by mAbs to LFA-3 (CD58) and CD44, which are expressed by T cells and EC. mAb to ICAM-1 (CD54, a ligand for LFA-1) provides inconsistent inhibition, and mAb to ICAM-2, used with or without anti-ICAM-1, is not inhibitory. Because both of these mAb block adhesion of LFA-1 expressing T cells to EC, our data suggest that additional ligands for LFA-1 must be important for allogeneic proliferation. mAb to VLA-4 a or [beta] chains (CD49d, CD29) enhance proliferation, presumably through direct costimulation of the T cells by these antibodies. However, a mAb to VCAM-1, an EC ligand for VLA-4, is partially inhibitory. In summary, our data suggest that CD4+ T cells directly recognize allogeneic HLA-DR molecules on EC, and further interact with EC ligands via CD2, LFA-1, and CD44. EC LFA-3 appears to be an important costimulator of T cell responses, whereas interaction with EC ICAM-1, ICAM-2, and VCAM-1 appear to be less critical for induction of T cell responses.