In this study we assessed the viability of cultured human umbilical vein endothelial cells (HUVE) treated with bacterial lipopolysaccharide (LPS), recombinant human interleukin-1 (rhIL-1), or recombinant human tumor necrosis factor-α (rhTNF-α) during inhibition of RNA or protein synthesis. Cytotoxicity was determined by 51Cr activity retained in labeled HUVE monolayers after exposure to LPS, rhIL-1 or rhTNF-α, and cycloheximide (Cx) or actinomycin D (Act D). Lipopolysaccharide (150 ng/ml), rhIL-1 (100 pg/ml), or rhTNF-α (20 ng/ml) alone was not toxic to HUVE in an 18-hr incubation. Cycloheximide alone (1 μg/ml for 18 hr) or Act D alone (1 μg/ml for 6 hr) was also not toxic to HUVE. However, coincubation of HUVE with Cx and LPS (150 ng/ml), rhIL-1 (10 pg/ml), or rhTNF-α (20 ng/ml) produced significant cytotoxicity at 18 hr (70 ± 4% for LPS, 75 ± 5% for rhIL-1, and 52 ± 5% for rhTNF-α; mean ± SEM of 18, 16, and 19 separate experiments, respectively). Similarly, coincubation of HUVE with Act D and LPS, rhIL-1, or rhTNF-α resulted in 82 ± 5%, 85 ± 3%, and 67 ± 4% cytotoxicity, respectively, at 6 hr (mean ± SEM of 5 separate experiments for LPS, and 7 separate experiments each for rhIL-1 and rhTNF-α). At the highest concentrations of LPS, rhIL-1, or rhTNF-α, cytotoxicity during coincubation with Cx or Act D was detected as early as 2 hr and was near maximal by 6 hr. In contrast to LPS, rhIL-1, or rhTNF-α, recombinant human interferon-γ (up to 100 U/ml), or human α-thrombin (up to 10 U/ml), produced no cytotoxicity in the presence of Cx. Recombinant human lymphotoxin (up to 50 ng/ml) had a detectable cytotoxic effect in the presence of Cx although it was significantly less than that seen with rhTNF-α. Furthermore, coincubation of human fibroblasts and human smooth muscle cells with Cx and LPS, rhIL-1, or rhTNF-α produced no cytotoxicity. We conclude that under these culture conditions, LPS, rhIL-1, or rhTNF-α produces a lethal injury to HUVE when de novo RNA or protein synthesis is inhibited. These results suggest that LPS, rhIL-1, and rhTNF-α may act via a common pathway in endothelial cells and that protein synthesis is important in regulating the response to these Stimuli.