Animal models of diabetes mellitus during pregnancy have repeatedly suggested that maternal hyperglycemia was teratogenic during organogenesis, and thus may contribute to diabetic teratogenesis. However, little attention has been focused on the effects of hyperglycemia on pre-organogenic development. In this report, we examine the effect of hyperglycemia (950 mg glucose/dL) on the development of mouse pre-embryos in vitro. B6C3F1 mice were superovulated with 5 U pregnant mare serum gonadotropin (PMSG) followed by 5 U human chorionic gonadotropin (hCG) 48 hours later. Two cell pre-embryos were recovered 48 hours later, pooled together, and randomly assigned to different treatment groups. Cultures were performed in HAM's F-10 media (Gibco, Long Island, NY) with 0.1% bovine serum albumin (BSA; Sigma, St. Louis, MO) BSA at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 with 15 to 30 embryos per milliliter of culture fluid. Cultures were viewed daily at 24, 48, and 72 hours after culturing, with recording of the development. Compared with control pre-embryos (n = 216), embryos cultured in elevated glucose levels (950 mg/dL) (n = 226) demonstrated marked growth retardation as assessed both by (1) distribution of developmental stages at each observation point (24 hours, P < .001; 48 hours, P < .006; 72 hours, P < .001); and (2) a difference in the average rank sums indicating a delay in maturation (P < .005). In a second protocol group, pre-embryos were cultured in an equivalent amount of l-glucose; no impairment in development compared with controls was noted. Additionally, the observed effects of d-glucose were not prevented by the addition of arachidonic acid (80 μg), in contrast to findings during organogenesis in the rat. We conclude that hyperglycemia per se exerts a deleterious effect on mouse pre-embryo development. This observation thus extends previous observations of glucose-induced embryopathy beginning during the period of organogenesis, to illustrate that the vulnerability to hyperglycemic insults begins even earlier in gestation.