Early compacting mouse morulae were placed in alpha-amanitin at various times after HCG and the effect of this transcriptional inhibitor during formation of the blastocyst was noted. No cavitation was observed in those embryos transferred into alpha-amanitin before 77 h after HCG but the percentage of cavitation increased dramatically in those embryos transferred after 80 h post HCG. The large increase in amino acid incorporation which normally occurs during fomration of the blastocyst was blocked in those embryos placed in the inhibitor before 80 h post HCG. Two-dimensional polyacrylamide gel electrophoresis of radiolabelled embryos showed that the changes in synthesis of certain polypeptides normally associated with blastocyst formation did not occur in those embryos placed in alpha-amanitin before 80 h after HCG. Only one cleavage division appears to occur in the presence of alpha-amanitin. However, groups of embryos which had completed their fifth division before exposure to the drug subsequently were able to form blastocysts and showed the quantitative and qualitative changes associated with blastocyst formation despite the continued presence of the inhibitor. These results suggest that a critical transcriptional event concerned with blastocyst formation occurs around 80 h after HCG and may be associated with the fifth cleavage division.