The interleukin-1β converting enzyme (ICE) is the cysteine proteinase responsible for cleaving the 31-kDa interleukin-1β (IL-1β) precursor to its active 17-kDa form. In lipopolysaccharide-stimulated cultured macrophages, induction of apoptosis but not necrosis effectively induces conversion of the IL-1β precursor to its mature form and results in the concomitant release of the mature cytokine from the cell. To determine whether ICE activity is required for macrophage apoptosis, we have exposed macrophages either to 5 mM ATP or to alloreactive cytolytic T lymphocytes (CTL) in the absence and presence of the ICE inhibitor peptide YVAD- chloromethylketone (YVAD-cmk). Activated cells treated with YVAD-cmk and ATP or CTL showed no mature IL-1β in either the cell lysates or the culture supernatants, indicating effective inhibition of ICE activity; however, the YVAD-treated macrophages showed no detectable change in 51Cr release or nuclear fragmentation, indicating failure to inhibit apoptotic cell death. Thus, in these cells, YVAD-cmk uncouples IL-1β processing and apoptosis.