Decreased responsiveness of the vasculature to vasoconstrictors has been implicated in the pathogenesis of endotoxic shock, yet the mechanism of diminished responsiveness has not been determined. In these studies, exposure of rat aortic rings to purified Escherichia coli lipopolysaccharide (endotoxin) in vitro inhibited subsequent contractions caused by vasoconstrictors. Contractions caused by the alpha-adrenoceptor agonist phenylephrine, as well as those induced by potassium depolarization, were depressed by endotoxin. The effect of endotoxin on vascular contractions was delayed. Phenylephrine-induced contractions were not decreased during a 1-h exposure to endotoxin (10 micrograms/ml), but they were markedly decreased when tested several hours after the exposure period. A large part of the inhibition caused by a 1-h exposure to endotoxin was endothelium dependent. In contrast, endotoxin inhibited contractions equally in rings with or without endothelium exposed to endotoxin for a longer period (3 h). The inhibitory effect of endotoxin was not affected by indomethacin, but it was eliminated in aortic rings treated with the protein synthesis inhibitor cycloheximide. These studies indicate that endotoxin potently inhibits vascular contraction in vitro. The effect of endotoxin is apparently independent of prostanoids but may involve protein synthesis and effects on both vascular smooth muscle and endothelial cells.