Activation of human T cells requires presentation of antigen by Ia (HLA-DR in man) bearing cells of the mononuclear phagocytic series (macrophages, MO, and more recently Langerhans cells, dendritic cells, and vascular endothelial cells. Since T cells must cross endothelial barriers to enter extravascular tissues during immune reactions, we investigated the role of endothelial cells in antigen presentation. Endothelial cells were cultured from human umbilical veins and identified by classic morphology and specific markers (factor VIII related antigen, and so on). Antigen-pulsed endothelial cells were used to present antigen to MO-depleted human T cells; activation was assessed by 3H-thymidine uptake. The HLA-DR compatible endothelial cells were as effective as MO in reconstituting MO-depleted T-cell responses. The endothelial cell reconstituted responses were antigen specific, HLA-DR restricted, and blocked by monoclonal antibodies to HLA-DR framework structures. Moreover, the T-cell responses were clonal with respect to HLA-DR. A monoclonal antibody completely eliminated MO reconstitution of the MO-depleted response without diminution of endothelial cell reconstitution of the same response. Fibroblasts and smooth muscle cells cultured from the same umbilical veins could not reconstitute the MO-depleted T-cell response. These data indicate that endothelial cells play an important and distinctive role in lymphocyte triggering.