Phosphatidylserine (PS) is a quantitatively minor, but physiologically important, phospholipid in mammalian cells. PS is synthesized by two distinct base-exchange enzymes, PS synthase-1 (PSS1) and PS synthase-2 (PSS2), that are encoded by different genes. PSS1 exchanges serine for choline of phosphatidylcholine whereas PSS2 exchanges ethanolamine of phosphatidylethanolamine for serine. We previously generated mice lacking PSS2 [Bergo, M.O. et al. J. Biol. Chem. (2002) 277:47701] and found that PSS2 is not required for mouse viability. We have now generated PSS1-deficient mice. In light of the markedly impaired survival of Chinese hamster ovary cells lacking PSS1 we were surprised that PSS1-deficient mice were viable, fertile, and had a normal life span. Total serine-exchange activity (contributed by PSS1 and PSS2) in tissues of Pss1-/- mice was reduced by up to 85% but, except in liver, the PS content was unaltered. Despite the presumed importance of PS in the nervous system, the rate of axonal extension of PSS1-deficient neurons was normal. Inter-crosses of Pss1-/- mice and Pss2-/- mice yielded mice with three disrupted Pss alleles but no double knock-out mice. In Pss1-/-/Pss2+/- and Pss1+/-/Pss2-/- mice, serine-exchange activity was reduced by 65-91% and the tissue content of PS and phosphatidylethanolamine was also decreased. We conclude that (i) elimination of either PSS1 or PSS2, but not both, is compatible with mouse viability; (ii) mice can tolerate as little as 10% of normal total serine-exchange activity, (iii) mice survive with significantly reduced PS and PE content.