ProIL-1 beta processing by IL-1 beta-converting enzyme (ICE) and the subsequent release of mature IL-1 beta are highly regulated events in the monocyte/macrophage response to pathogens. This process occurs in a controlled way through the activation of the constitutively expressed 45-kDa ICE precursor (proICE). To characterize the signaling pathways involved in ICE regulation in human monocytes/macrophages, we analyzed ICE activation in the presence of specific inhibitors of classic signaling pathways. Although LPS-induced ICE activity was not significantly affected by interruption of extracellular signal-regulated kinase, p38 kinase, or phosphoinositol 3-kinase, Janus kinase 3 (JAK3) inhibition produced a significant dose-dependent enhancement of LPS-induced ICE activity. Support for the inhibitory role of JAK3 was shown by the fact that IL-4 (which uses JAK1 and JAK3 signaling) suppressed LPS-induced ICE activity and by the finding that JAK3 knockout macrophages have increased LPS-induced ICE activation. To understand how JAK3 down-regulates LPS-induced ICE activity in monocytes, we hypothesized that JAK3 signaling enhances IL-10 production. In support of this model we show that LPS-induced IL-10 expression was synchronous with ICE deactivation, IL-4 induced the release of IL-10, exogenous IL-10 suppressed LPS-induced ICE activity, a neutralizing IL-10 Ab increased LPS-induced ICE activity, and, finally, JAK3 knockout macrophages displayed significantly reduced LPS-induced IL-10 production. These findings support a model in which JAK3 signaling enhances IL-10 production leading to down-regulation of ICE activation and suppression of IL-1 beta processing and release.