Fas-mediated apoptosis has been shown to be mediated by the IL-1 β converting enzyme (ICE) pathway. To determine the relationship between ICE and its substrate IL-1β, we examined six human cell lines for susceptibility to Fas-mediated apoptosis and Fas induction of ICE-like activity. The human B lymphoblastoid cell line SKW6.4 and the human T lymphoma cell lines Jurkat, CEM-6, H-9, and MOLT4 were susceptible to Fas-mediated apoptosis, whereas the human promyelocytic leukemia cell line HL-60 was resistant to Fas-mediated apoptosis. ICE mRNA was highly expressed in SKW6.4, H-9, and HL-60 cells, and ICE-like activity increased during Fas-mediated apoptosis in SKW6.4 cells. In contrast, IL-1β mRNA was highly expressed only in HL-60 cells. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone, a tetrapeptidyl inhibitor of ICE, prevented Fas-mediated apoptosis strongly in SKW6.4 and H-9 cells but weakly or marginally in other cells. To examine whether intracellular IL-1β is a proteolytic substrate or an endogenous competitive inhibitor against other substrates for Fas-ICE-mediated apoptosis in SKW6.4 cells, we established precursor IL-1β transfectant clones using SKW6.4 cells. We demonstrated that stably transfected SKW6.4 cells expressing precursor IL-1β, but not cells transfected with the empty vector, exhibited resistance to Fas-mediated apoptosis due to competitive inhibition of ICE-like activity, which was associated with increased cleavage of precursor IL-1β to mature IL-1β. These results suggest that Fas-mediated apoptosis is mediated by ICE cleavage of proteolytic substrates other than IL-1β and that IL-1β is an endogenous inhibitor of Fas-mediated apoptosis.