Cultured human endothelial cells (EC) resist tumor necrosis factor (TNF)-mediated apoptosis. However, the combination of TNF and the protein synthesis inhibitor cycloheximide (CHX) induces apoptosis in up to 50% of EC within 24 hours. TNF + CHX killing is effectively blocked by transfected CrmA protein or treatment with Z-VAD.fmk peptide-both inhibitors of interleukin-1-converting enzyme-like proteases-but not by transfected antiapoptotic proteins Bcl-2, Bcl-XL, or A1. C6-ceramide (cer) can also sensitize EC to TNF-induced apoptosis. TNF + cer killing, which can affect more than 50% of EC, is not effectively inhibited by CrmA or Z-VAD frank, but can be readily blocked by Bcl-2, Bcl-XL, or A1. Both TNF + CHX and TNF+ cer killing are induced by a TNF mutein that only interacts with the type 1 TNF receptor, and both responses can be inhibited by the antiapoptotic protein A20. These data suggest that TNF activates two biochemically separable pathways of EC injury.