Objective:To determine whether human vascular endothelial cells produce tumor necrosis factor-α (TNF-α) after stimulation with proinflammatory cytokines and bacterial lipopolysaccharides (LPS).Design:Prospective,in vitrorepeated-measurements analysis of cellular responses.Setting:Research laboratory in an academic medical center.Subjects:Human umbilical vein endothelial cells (HUVECs).Interventions:HUVECs were incubated with interferon-γ (IFN-γ), interleukin-1β (IL-1β), and LPS, or their different combinations for 2 to 48 hrs.Measurements and Main Results:TNF-α was measured by time-resolved immunofluorometric assay. Unstimulated HUVECs did not produce detectable amounts of TNF-α, but IFN-γ, IL-1β, and LPS when added together induced TNF-α production of HUVECs in a time-dependent manner. Immunofluorescent staining confirmed that the TNF-α was produced by endothelial cells. IFN-γ, IL-1β, or LPS alone did not induce TNF-α production, whereas IFN-γ and IL-1β in combination were able to induce TNF-α production to some extent, and the production could be further increased with LPS. TNF-α messenger RNA expression was detected with reverse transcriptase-coupled polymerase chain reaction in stimulated, but not in unstimulated, HUVECs.Conclusions:HUVECs are capable of producing TNF-α after proinflammatory cytokine stimulation and may therefore contribute to the increased amount of TNF-α found in pathologic states such as septic shock.