It has been suggested that tumor necrosis factor alpha (TNF alpha) acts not only by direct toxicity, but also as a proximal mediator which is able to induce the production of other cytokines, especially interleukin 6 (IL-6) and interleukin 1 beta (IL-1 beta). In order to test the dependence of the release of these two cytokines from leukocytes upon induction by TNF alpha, we stimulated whole blood in vitro with TNF alpha and compared the cytokine levels with those induced by endotoxin. The cytokine release was also determined after stimulation by endotoxin with added TNF alpha and by endotoxin with monoclonal antibodies against TNF alpha (anti-TNF alpha) added in order to reduce TNF alpha. Unstimulated blood samples were used as controls. The plasma levels of both IL-6 and IL-1 beta were significantly higher after stimulation with endotoxin than after stimulation with TNF alpha. TNF alpha did not induce cytokine levels significantly higher than controls. The cytokine levels were the same whether or not anti-TNF alpha was included together with the endotoxin. Plasma from samples with added anti-TNF alpha had no detectable TNF alpha. Our results indicate that the leukocyte-derived production of IL-6 and IL-1 beta in whole blood is stimulated directly by endotoxin and is not mediated by TNF alpha.