Endotoxemia results in the release of cytokines that exert complex effects on the cardiovascular system. The purpose of this study was to 1) determine if interleukin 1 β (IL1 β) and tumor necrosis factor α (TNFα) elicit the release of endothelium-derived relaxing factor (EDRF) and nitric oxide derived from the constitutive nitric oxide synthase present in vascular endothelium, and 2) determine if these cytokines alter endotoxin-mediated decreases in EDRF bioactivity and nitric oxide production. Cultured bovine aortic endothelial cells were directly exposed to endotoxin, human recombinant TNFα, interleukin 1 β, or a combination of endotoxin and cytokine for 1 h, followed by a second hour without endotoxin. Subsequently, both basal as well as agonist-stimulated (bradykinin) EDRF bioactivity and nitric oxide (NO) content of the effluent were quantitated. In additional experiments, endothelial cells were exposed acutely over a 30-min assay period to either endotoxin alone, cytokine alone, or endotoxin and cytokine. Following the 2-h incubation, endotoxin alone markedly reduced basal EDRF bioactivity and NO production (44 ± 13% control, 66 ± 13% control, respectively) and decreased bradykinin-stimulated EDRF bioactivity and NO production (58 ± 5% control, 55 ± 4% control, respectively). TNFα and IL1 β did not stimulate EDRF release or NO production either acutely or after prolonged exposure, nor did they alter agonist-stimulated EDRF bioactivity and NO production. Similarly, co-incubation of endotoxin with TNFα or IL1 β failed to significantly alter the inhibitory effects of endotoxin on EDRF bioactivity and NO production. These results demonstrate that 1) the specific cytokines TNFα and IL1 β do not elicit the release of constitutively derived EDRF from endothelial cells, 2) endotoxin at low concentrations directly inhibits both EDRF bioactivity and NO production, and 3) the cytokines TNFα and IL1 β fail to alter the inhibitory effects of endotoxin on EDRF/NO synthesis from the constitutive NO synthase of vascular endothelial cells.