During the early stages of heart development, there are two main foci of cell death: in the outflow tract (OT) endocardial cushions and in the atrioventricular (AV) endocardial cushions. These tissues contribute to the septa and valves of the mature heart, and receive cell populations from neural crest (NC) cell migration and epicardial cell invasion. We examined embryonic chick hearts for expression, in the cushions, of bcl-2 family members, caspase-9, and the caspase substrate poly (ADP)-ribose polymerase (PARP). Anti-apoptotic bcl-2 is expressed heavily in the OT and AV regions, throughout ED 4-7, with a decrease in levels at ED 4 in the OT and ED 5 in the AV cushions. Pro-apoptotic bax predominantly associated with the prongs of the NC-derived aortico-pulmonary (AP) septum, but was expressed throughout the AV cushions. Pro-apoptotic bak also associated with the prongs of the AP septum in the OT, while protein levels were up-regulated at ED 4-5 in the OT and ED 4-6 in the AV cushions. Bid expression showed a similar time-course. We found the 10 kDa cleavage fragment of active caspase-9 from ED 4-8 in the OT and from ED 5-8 in the AV cushions, and the 24 kDa cleavage fragment of PARP throughout ED 3-8 in the OT and ED 7-8 in the AV cushions. Caspase-3 cleavage occurred throughout the time period examined. Using cushion cell cultures, we found that inhibitors of caspases-3 and -9 and a universal caspase inhibitor all significantly reduced apoptosis, as did retroviral over-expression of bcl-2 using an RCAS expression vector. Pre-migratory NC cells were fluorescently labeled in vivo with DiI. Subsequent nuclear staining of cushion cells with DAPI revealed the presence of apoptotic nuclei in the neural crest cells in the OT cushions, and in the prongs of the AP septum. These results demonstrate a develop-ment-ally-regulated role for the bcl-2 and the caspase families of molecules in the endocardial cushions of the developing heart, and lend support to the possibility that some of the dying cells in the cushions are neural crest-derived.