The diabetic state, as well as elevated culture media glucose level (950 mg d-glucose/dL) per se, significantly retards in vitro development of mouse pre-implantation embryos from a two-cell stage to blastocyst stage; maternal insulin therapy to diabetic mice reverses this impairment. This study was undertaken to assess (1) whether less extreme elevation of the media glucose concentration would also impair development, and (2) whether elevated culture media insulin or glucagon levels would alter development. Two-cell pre-embryos were recovered from B6C3F1 mice that had been stimulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hGG), mated, and killed 48 hours later. Pre-embryos were observed in culture at 24-hour intervals for a total of 72 hours at four glucose levels: 110 (n = 108), 220 (n = 101), 440 (n = 65), and 950 (n = 106) mg d-glucose/dL. Impairment in progression of development was noted at each time period; compared with development in 110 mg glucose/dL, the distribution of development was significantly different at 24 hours (χ2 = 60.1, P < .001), at 48 hours (χ2 = 36.7, P < .001), and at 72 hours (χ2 = 45.1, P < .001). Rate of development as assessed by ANOVA was also significantly reduced at increasing glucose levels (P < .0001), with Duncan Multiple Range test demonstrating differences between development at higher glucose levels in the comparison of development in 110 mg/dL versus 440 mg/dL and 950 mg/dL, and at 220 mg/dL versus 950 mg/dL. Comparison of pre-embryo growth at different insulin concentrations, or in the presence of glucagon, showed no significant effect on pre-embryo growth and development compared with controls. We conclude that elevations of glucose to levels of 440 mg/dL can cause significant impairment of pre-embryo growth and development.