Activation of endothelial cells by endotoxin (lipopolysaccharide, LPS) may occur through two different pathways. LPS can directly activate endothelial cells through its interaction with soluble CD14 or indirectly via cytokines produced in blood in response to LPS. Substitution of whole blood for plasma apparently increases the endothelial cells responses to LPS by a factor of 1,000, rendering them sensitive to subpicomolar quantities of LPS. This shift in sensitivity is dependent on the presence of monocytes or conditioned plasma from whole blood incubated with small concentrations of LPS. Herein, using agents that block the effects of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), we demonstrate that TNF-alpha and IL-1 beta are the two LPS-induced cytokines responsible for the activation of endothelial cells, produced in blood in response to picomolar quantities of LPS. Anti-TNF-alpha monoclonal antibodies (mAbs) and IL-1 receptor antagonist separately had partial inhibitory effects. Complete and sustained inhibition of endothelial cell activation was obtained only when the two inhibitors were added together. We conclude that TNF-alpha and IL-1 beta induced in whole blood by picomolar concentrations of LPS mediate endothelial cell activation to these small quantities of LPS and that blocking of both cytokines is necessary to inhibit LPS-induced blood-dependent endothelial cell activation.