The development and resolution of an inflammatory process are regulated by a complex interplay among cytokines that have pro-and anti-inflammatory effects. Effective and sustained action of a proinflammatory cytokine depends on synergy with other inflammatory cytokines and antagonism of opposing cytokines that are often highly expressed at inflammatory sites. We analyzed the effects of the inflammatory and stress agents, IL-1, TNF-α, LPS, sorbitol, and H 2 O 2, on signaling by IL-6 and IL-10, pleiotropic cytokines that activate the Jak-Stat signaling pathway and have both pro-and anti-inflammatory actions. IL-1, TNF-α, and LPS blocked the activation of Stat DNA binding and tyrosine phosphorylation by IL-6 and IL-10, but not by IFN-γ, in primary macrophages. Inhibition of Stat activation correlated with inhibition of expression of IL-6-inducible genes. The inhibition was rapid and independent of de novo gene induction and occurred when the expression of suppressor of cytokine synthesis-3 was blocked. Inhibition of IL-6 signaling was mediated by the p38 subfamily of stress-activated protein kinases. Jak1 was inhibited at the level of tyrosine phosphorylation, indicating that inhibition occurred at least in part upstream of Stats in the Jak-Stat pathway. Experiments using Stat3 mutated at serine 727 and using truncated IL-6Rs suggested that the target of inhibition is contained within the membrane-proximal region of the cytoplasmic domain of the gp130 subunit of the IL-6 receptor and is different from the SH2 domain-containing protein-tyrosine phosphatase/suppressor of cytokine synthesis-3 docking site. These results identify a new level at which IL-1 and TNF-α modulate signaling by pleiotropic cytokines such as IL-6 and IL-10 and provide a molecular basis for the previously described antagonism of certain IL-6 actions by IL-1.