Background: Apoptosis plays an important role in the normal development and homeostasis of metazoans. Aberrations in this process have been implicated in several major human diseases, but its molecular mechanism is poorly understood. In animals as diverse as humans and nematodes, the Bcl-2 oncoprotein prevents or delays apoptosis, whereas proteases of the interleukin-1 β-converting enzyme (ICE) family are required, suggesting that they are components of a conserved mechanism controlling the onset of apoptosis. Results: A cell-free system produced from Xenopus laevis eggs reproduces nuclear events characteristic of apoptosis after a lag phase. We have used this system to define the temporal sequence of biochemical events and to examine the relationship between Bcl-2 and apoptotic proteases. Bcl-2 prevents apoptotic chromatin condensation and DNA cleavage, but only when added prior to the activation of a protease which has characteristics similar to the Ced-3 sub-family of ICE-like proteases and which cleaves poly(ADP-ribose) polymerase (PARP). Bcl-2 attenuates activation of this protease, an effect that does not require de novo protein synthesis or the presence of intact nuclei. The Cap-3-related protease CPP-32 is cleaved during the late stages of apoptosis in this system and after PARP cleavage. Generation of CPP-32-cleaving activity is inhibited by Bcl-2. Conclusions: These experiments provide direct biochemical evidence that Bcl-2 protects against apoptosis, at least in part, by regulating the activation of a series of apoptotic proteases that cleave PARP and other substrates. This cell-free system provides a useful biochemical model for analyzing the molecular mechanism controlling the activation of these proteases.