Administration of lipopolysaccharide (LPS) results in activation of the hypothalamic-pituitary-adrenal axis. LPS induces the release of a number of proinflammatory cytokines, i.e. interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF), which activate the hypothalamic-pituitary-adrenal axis as well and may mediate the effects of LPS. Variations in the kinetics of appearance of IL-1, TNF, and IL-6 after LPS challenge suggested that these cytokines may play different roles at different times. To elucidate the mutual dependence and contribution of individual cytokines in the course of LPS-induced ACTH release, we used blocking antibodies to IL-6, TNF, and the IL-1 receptor. Our results demonstrate that anti-IL-6 antibody abrogated ACTH induction throughout the course of the response both 2 and 4 h after LPS challenge. In contrast, anti-IL-1 receptor and anti-TNF antibody, given individually, blocked ACTH production at 4 h, but not at 2 h. Only combined administration of these two antibodies diminished, but did not eliminate, ACTH release at 2 h. This is the first demonstration that all three inflammatory cytokines are obligatory for LPS-induced elevation of plasma ACTH. In addition, these results suggest that IL-1, IL-6, and TNF play different roles in LPS-induced ACTH release.