The effect of choline deprivation on the fatty acid composition of liver phospholipids was determined in male rats fed the test diet for 3 weeks in one experiment and for 2 weeks in another experiment.Phospholipids from total lipid extracts were precipitated in acetone saturated with MgCl2, and their acyl groups examined by gas chromatography. The concentration of arachidonic acid in the total phospholipids was inversely related to the total amount of lipid accumulated in the liver. Analyses of fatty acids released by snake venom also demonstrated the reduced proportion of arachidonic acid in the fatty livers of choline-deprived rats. In deficient livers having relatively low lipid deposits, there were increased concentrations of docosapentaenoic and docosahexaenoic acids. Choline deprivation appeared not to alter the fatty acid composition of phosphatidylcholine, but less of this phospholipid was synthesized. The most striking effect of the deficiency was the decreased concentration of arachidonic acid compensated by more unsaturated acids in liver phosphatidylethanolamine.