The role and mechanism of cell death in early mammalian embryos is not well understood. In mouse embryos collected after fertilization and maintained in vitro until blastula formation, two instances of cell death are observed: the polar bodies and one or two cells near the equator, at the junction of the inner cell mass to the prototrophoblast. Inhibitors of caspases do not block the death of the polar bodies. Inhibitors of caspases 3, 7 and 8 do not affect post-cavitation death, but the pan-caspase inhibitor zVAD-FMK, when applied at the 1-2 cell stage, causes an expansion of post-cavitation death and ultimately malformation or death of the embryo. Our results indicate that the early deaths are not caspase-dependent and that there is a role for caspase activity in early embryos, which is not related to cell death. The caspase cascade is a major component of apoptosis in development and in many other situations. Presently most researchers assume that caspases function only in cell death, even though related family members serve, for instance, in the activation pathway for immunologically competent cells. If caspases have other roles in cell function, it is appropriate to ask what roles they play in early development. There have been a number of reports on cell death in pre-implantation embryos. The polar bodies begin to die during earliest cleavage, but cell death in the pre-implantation embryo itself begins at about the 8-cell stage. The cell death in the embryo is apoptotic by the criterion of TUNEL positivity (Matwee, Betts and King, 2000). The expression of caspases 2 and 3 (but not caspase 1) has been shown for blastocysts (Weil, Jacobson, Coles, Davies, Gardner, Raff and Raff, 1996). A few cells routinely die in the blastocyst (Handyside and Hunter, 1986) (Weil, Jacobson, Coles, Davies, Gardner, Raff and Raff, 1996). However, pre-implantation development is not disrupted by individually knocking out Bcl-2, Bax, Bcl-X, Bcl-W, or caspases 1, 2,3,8,9, or 11 (Pampfer and Donnay, 1999).During embryonic development of mammals, zygotic genes are translated as early as the 2-cell stage. The first sign of cell death is seen at the 6-8 cell stage, followed by further deaths at the blastula stage. It is believed that cell death in these stages relates to cavitation and blastocyst formation and the elimination of potential trophectoderm cells from the inner cell mass (Pierce, Lewellyn and Parchment, 1989). Although cavitation occurs synchronously with cell death, at least in embryoid bodies, this death requires apoptosis-inducing factor but is independent of caspase expression and does not manifest all the characteristics of apoptosis (Joza, Susin, Daugas, Stanford, Cho, Li, Sasaki, Elia, Cheng, Ravagnan, Ferri, Zamzami, Wakeham, Hakem, Yoshida, Kong, Mak, Zuniga-Pflucker, Kroemer and Penninger, 2001).To determine if specific caspases have different roles in the different stages of preimplantation mouse embryos, we exposed the developing embryos to known caspase inhibitors, the fluoromethylketone (FMK) deriva...