The mechanism by which p53 modulates apoptosis in cancer therapy is incompletely understood. Here, cell-free extracts from irradiated tumor cells are described in which endogenous p53 protein is shown to participate in caspase activation. This apoptotic activity is also oncogene-dependent, but independent of transcription in general or the presence of Bax or cytochrome c. A general use for this system is as a cell-free screen for apoptosis modulators. In this way, profound effects of protein kinase A were identified and corroborated in vivo by the protection conferred by cAMP against diverse triggers of p53-dependent apoptosis. This system provides direct biochemical evidence that p53 protein can transduce apoptotic signals through protein- protein interactions and reveals a modulator kinase pathway capable of regulating p53-dependent caspase activation.