A single-gene nuclear choline-requiring mutant of Saccharomyces cerevisiae was studied. Choline as a growth supplement to synthetic media could be substituted by low concentrations of dimethylethanolamine, monomethylethanolamine or ethanolamine. DL-Serine also supported growth, but only at high concentrations: on a molar basis it was approximately one hundred times less effective than choline. When cultured in unsupplemented medium the mutant cells soon ceased to grow. The growth-arrested cells contained less than one fifth of the phosphatidylethanolamine present in wild-type cells and only traces of phosphatidylserine. The relative content of the two phospholipid species was raised by growing the mutant cells in the presence of choline of the other supplements but still remained lower than in wild-type cells. The mutant cells depleted of phosphatidylethanolamine and phosphatidylserine had greatly diminished ability to fuse with other cells in mating and their protoplasts showed increased resistance to hypotonic lysis. Respiration was not substantially affected by the deficit of the two phospholipid species in the mutant. In cell-free preparations, the affinity of the phosphatidylserine synthesizing system for serine was found to be almost two orders of magnitude lower in the mutant than in the wild-type. The impairment of phosphatidylserine synthesis accounts for growth requirement and the abnormal phospholipid composition of the mutant cells.