Recent studies have yielded contradictory interpretations about the influence of gram-negative endotoxin on endothelium-derived relaxing factor (EDRF). We tested the hypothesis that Escherichia coli endotoxin exerts primary facilitatory or, alternatively, inhibitory actions on EDRF release and the synthesis of either nitric oxide or a nitroso compound in cultured endothelial cells. Bovine aortic endothelial cells were grown on microcarrier beads and either exposed acutely (30 min) to E. coli endotoxin or incubated with endotoxin for 1 h followed by a 1-h wash (prolonged exposure). EDRF bioactivity was measured under basal, bradykinin-stimulated, and A23187-stimulated conditions using standard isometric tension recordings. EDRF-derived nitric oxide was quantitated using a specific chemiluminescence technique. Endotoxin (0.005-5 micrograms/ml) decreased EDRF bioactivity and nitric oxide production under both basal and bradykinin-stimulated conditions after prolonged, but not acute, exposure. A23187-stimulated EDRF bioactivity and nitric oxide production were minimally, albeit significantly, reduced after endotoxin. The present results demonstrate that EDRF activity and nitric oxide production are decreased in vascular endothelial cells exposed to endotoxin. Endotoxin itself failed to directly stimulate EDRF release from endothelium. Alternative sources of nitrovasodilators, endothelium-independent effects, or release of other vasoactive mediators by endotoxin may be responsible for systemic hypotension during in vivo endotoxemia.