Endothelium is an important target of tumor necrosis factor/cachectin (TNF), a central mediator of the host response in endotoxemia and Gram-negative sepsis. In this report, TNF is shown to increase the permeability of endothelial cell monolayers to macromolecules and lower molecular weight solutes by a mechanism involving a pertussis toxin-sensitive regulatory G protein. Within 1-3 h of exposure to TNF (5 nM), changes in cell shape/cytoskeleton occurred that led to disruption of monolayer continuity with the formation of intercellular gaps. Correlated with these structural changes was an increase in endothelial permeability to macromolecular and lower molecular weight tracers; time-dependent, reversible increases in passage of these tracers, evident by 1-3 h, were observed after addition of TNF to cultures. Perturbation of barrier function by TNF also depended on the dose of TNF added being half-maximal by approximately 0.4 nM. Only a brief exposure (15 min) of TNF to endothelium was required to induce an increase in permeability, and this was not prevented by the presence of cycloheximide or actinomycin D. Preincubation of monolayers with pertussis toxin blocked in parallel TNF-induced increased passage of solutes and cell shape/cytoskeletal perturbation, indicating the close correlation between these changes in endothelial cell function. In contrast, pertussis toxin did not alter TNF-induced modulation of two endothelial cell coagulant properties. These data provide evidence for two intracellular pathways of TNF action that are distinguishable by pertussis toxin and provide insight into a mechanism underlying loss of solute from the intravascular space mediated by TNF: alteration in endothelial cell barrier function.