AbstractSeveral thousand two‐cell mouse ova were cultivatedin vitro, and the effect on development of changing the osmolarity and hydrogen ion concentration of the medium was examined. Statistical experimental design techniques were employed throughout the experiments in order to make the most efficient use of the material available. The two‐cell ova were cultivated for three days, and the percentage which developed into normal blastocysts was used as a measure of the treatment effect.Changing the osmolarity of the medium had a marked effect on the number of blastocysts developing from two‐cell mouse ova. Blastocysts developed in media with osmolarities ranging between 0.2002 and 0.3542 osmols. The optimum osmolarity for the development of two‐cell mouse ova into blastocysts was 0.2760 osmols.The hydrogen ion concentration of the culture medium also had an effect on the development of two‐cell mouse ova into blastocysts. Although blastocysts developed in media with pH's ranging from 5.87 to 7.78, the optimum pH appeared to be