Phosphatidylserine (PS) in mammalian cells is synthesized through the exchange of free L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine (PE). The serine base exchange in Chinese hamster ovary (CHO) cells is catalyzed by at least two enzymes, PS synthase (PSS) I and II. A PSS I-lacking mutant of CHO-K1 cells, PSA-3, which exhibits approximately 2-fold lower serine base exchange activity than CHO-K1, is defective in the conversion of phosphatidylcholine to PS but has the ability to convert PE to PS. The PSA-3 mutant requires exogenous PS or PE for cell growth. In the present study, from PSA-3 mutant cells, we isolated a mutant, named PSB-2, with a further decrease in the serine base exchange activity. The activity in the homogenate of PSB-2 mutant cells was approximately 10% that of PSA-3 mutant cells and approximately 5% that of CHO-K1 cells. The PSB-2 mutant exhibited an approximately 80% reduction in the PSS II mRNA level relative to that in PSA-3 mutant and CHO-K1 cells. These results showed that the PSB-2 mutant is defective in PSS II. Like the PSA-3 mutant, the PSB-2 mutant grew well in medium supplemented with PS. However, in the medium supplemented with PE, the PSB-2 mutant was incapable of growth, in contrast to the PSA-3 mutant. In the medium with exogenous PE, the PSB-2 mutant was defective in PS biosynthesis, whereas the PSA-3 mutant synthesized a normal amount of PS. A metabolic labeling experiment with exogenous [32P]PE revealed that the PSB-2 mutant was defective in the conversion of exogenous PE to PS. This defect and the growth and PS biosynthetic defects of the PSB-2 mutant cultivated with exogenous PE were complemented by the PSS II cDNA. In addition, the cDNA of the other PS synthase, PSS I, was shown not to complement the defect in the conversion of exogenous PE to PS of the PSB-2 mutant, implying that PSS I negligibly contributes to the conversion of PE to PS in CHO-K1 cells. These results indicated that PSS II is critical for the growth and PS biosynthesis of PSA-3 mutant cells cultivated with exogenous PE and suggested that most of the PS formation from PE in CHO-K1 cells is catalyzed by PSS II.