RECENT studies suggest that proteases of the interleukin 1-beta-converting enzyme (ICE)/ced-3 family are involved in initiating the active phase of apoptosis(1-3). Here we identify a novel protease resembling ICE (prICE) that is active in a cell-free system that reproduces the morphological and biochemical events of apoptosis(4). prICE cleaves the nuclear enzyme poly(ADP-ribose) polymerase (PARP) at a tetrapeptide sequence identical to one of two ICE sites in pro-interleukin-1-beta. However, prICE does not cleave purified pro-interleukin-1-beta, and purified ICE does not cleave PARP, indicating that the two activities are distinct. Inhibition of prICE abolishes all manifestations of apoptosis in the extracts including morphological changes, cleavage of PARP and production of an oligonucleosomal ladder. These studies suggest that prICE might be pivotal in initiating the active phase of apoptosis in vitro and in intact cells.