AbstractThere has been considerable discussion as to whether the developing mouse ova requires an exogenous protein supply. A series of experiments were performed to determine the effect of various fixed‐nitrogen sources on the developmentin vitroof two‐cell mouse ova into blastocysts. It was found that two‐cell mouse ova would not develop into blastocysts in medium that did not contain a fixed‐nitrogen source. Crystalline bovine serum albumin (BSA) at 1 mg/ml or, alternatively, the constituent amino acids of BSA could supply this requirement for fixed‐nitrogen. In the presence of single amino acids at 10−3Mor 8 × 10−3M, only an occasional two‐cell ova developed into a blastocyst.Single amino acids were omitted from the medium containing the constituent amino acids of BSA in an attempt to demonstrate an essential amino acid requirement of two‐cell mouse ova. The omission of no single amino acid from the medium, completely prevented the development of some two‐cell ova into blastocysts. The only amino acid whose omission resulted in a significant decrease in development was cystine. In the medium where amino acids were used in place of BSA, a non‐protein polymer was used to replace the physical properties of albumin. The non‐protein polymers which appeared best were Polyvinylpyrrolidone (MW = 150,000), Aca