Background and Purpose: Pyruvate, Lactate and glucose have been used as energy substrates in most pre implantation embryo culture media. The individual and in combined form, roles of these substrates on preimplantation mouse embryo development in vitro were investigated.Materials and Methods: Two cell embryos, stimulated by ovulation in mouse were collected from the uterus by flashing method. Two cell embryos. Were collected by flashing using hepes buffered T6 (without any energetic substrate) 36 hours after injection of HCG. Embryos were cultured in seven media as follow.T6 medium containing pyruvate lactate and glucose (pL G), T6 medium containing pyruvate and lactate (pL), T6 medium Containing pyruvate and glucose (LG), T6 medium Containing pyruvate (P), T6 medium containing (L) and T6 medium containing glucose (G) the embryos were observed every 24 hours for the days.Results: The percentages of embryos reached compact morula stage were 78.12, 90, 69, 17, 91.45, 43.41 and 40 for pLG, pL.p G, LG, p and G respectively. No Significant difference was observed between pL G, pL, pG and p but there was significant differences between L, LG and G compare to the other groups, with lower percentages of reaching to morula stage (P<0.05).After 79 hours, 68.19, 79.23, 68.42, 41.08, 76.06, 11.62 and 18.51 percents of embryos reached blastocyst stage.Significant differences were observed between L, LG and G media Compare to the other media (P<0.05).Conclusion: The data suggest that only pyruvate can complete preimplantation development in vitro. Also these date do not confirm the synergistic effect of pyruvate, lactate and glucose in the support of embryo development.