To study the role of the yolk sac and amnion in craniofacial development, the effects of opening the yolk sac and amnion on facial formation of rat embryos were examined in vitro. Rat embryos were cultured for 72 hr from day 11.5 of gestation using an improved rotation apparatus. In experiments, the yolk sac and amnion were opened at the time of explantation (day 11.5) in one group (D11 open) and were opened 24 hr after the beginning of the culture (day 12.5) in another group (D12 open). Cleft lip developed in 100% of cultured embryos when the yolk sac and amnion were opened at day 11.5 (D11 open). In the D12 open group, however, cleft lip occurrence decreased to 3.0%. Protein content, wet weight, and somite number of cultured embryos were not significantly different in the two groups. The results of this study demonstrate that it is beneficial to open the yolk sac and amnion after 24 hr in culture for normal facial formation of rat embryo cultured from day 11.5 of gestation.