Many cells die during mammalian development and are engulfed by macrophages. In DNase II(-/-) embryos, the TUNEL-positive DNA of apoptotic cells is left undigested in macrophages, providing a system for studying programmed cell death during mouse development. Here, we showed that an Apaf-1-null mutation in the DNase II(-/-) embryos greatly reduced the number of macrophages carrying DNA at E11.5. However, at later stages of the embryogenesis, a significant number of macrophages carrying undigested DNA were present in Apaf-1(-/-) embryos, indicating that cells died and were engulfed in an Apaf-1-independent manner. In most tissues of the Apaf-1(-/-) embryos, no processed caspase-3 was detected, and the DNA of dead cells accumulated in the macrophages appeared intact. Many nonapoptotic dead cells were found in the tail of the Apaf-1(-/-) embryos, suggesting that the Apaf-1-independent programmed cell death occurred, and these dead cells were engulfed by macrophages. In contrast, active caspase-3 was detected in E14.5 thymus of Apaf-1(-/-) embryos. Treatment of fetal thymocytes with staurosporine, but not etoposide, induced processing of procaspases 3 and 9, indicating that the E14.5 thymocytes have the ability to undergo caspase-dependent apoptosis in an Apaf-1-independent manner. Thus, programmed cell death in mouse development, which normally proceeds in an efficient Apaf-1-depenent mechanism, appears to be backed up by Apaf-1-independent death systems.