In mammals, there are no reliable methods for synchronizing cell division of early embryos without reducing their ability to develop into blastocysts and fetuses. The present study was undertaken to examine the in vitro inhibition of cell division of four-cell mouse embryos by cell cycle arrest agents. The reversibility of the agents was also tested by examining the developmental ability of treated embryos. Four-cell mouse embryos obtained at 54 hr post-human chorionic gonadotrophin (post-hCG) were cultured for 4, 8, 12, or 16 h r in media supplemented with either nocodazole, a n inhibitor of tubulin polymerization, 6-dimethylaminopurine (6-DMAP1, a n inhibitor of maturation promoting factor (MPF) activation, or aphidicolin, a specific inhibitor of DNA polymerase alpha. Reversibility and toxicity of these agents were both dose and time dependent. For all three agents, prolonging cleavage arrest for 8 or 16 h r (at the effective concentrations) caused embryo lethality. Although nocodazole treatment was least cytotoxic, 6-DMAP and aphidicolin concentrations which induce cleavage arrest were detrimental to development beyond the blastocyst stage. The results of this study show that the development of embryos treated with these three cell-cycle-arrest agents is dose and incubation time dependent. Toxic effects beyond the blastocyst stage could only be minimized for nocodazole by reducing the exposure time of treatment and concentration of the mitotic inhibitor. However, these results render doubt on the usefulness of 6-DMAP and aphidicolin for synchronization studies leading to embryo transfer procedures.