A quantitative thin‐layer chromatographic method is described for the separation and isolation of subfractions with different degrees of unsaturation of phosphatidylcholines and phosphatidylethanolamines from rat liver. In fasting male and female rats the tetraunsaturated or arachidonoyl‐containing subfraction was found to be of quantitative dominance in both phosphatidylcholines and phosphatidylethanolamines, whereas the latter contained relatively more of the hexaunsaturated fraction than the phosphatidylcholines. All the monoenoic subfractions contained more palmitic acid than stearic acid. In both the phosphatidylcholines and the phosphatidylethanolamines more than 50% of the stearic acid in each phospholipid was recovered in the tetraenoic fraction in both male and female rats. Incorporation of 32P and [1,2‐14C2]ethanolamine was found to occur at a high rate in the hexaenoic phosphatidylethanolamines. Another characteristic finding was the comparatively low metabolic reactivity of the arachidonoyl fraction in both phosphatidylcholines and phosphatidylethanolamines. The results have tentatively been interpreted to reflect differences in substrate specificity of CDPcholine: 1,2‐diglyceride cholinephosphotransferase and CDPethanolamine: 1,2‐diglyceride ethanolaminephosphotransferase towards different molecular species of l‐1,2‐diacylglycerol. The differences observed between the hexa‐and tetraunsaturated subfractions may also be related to differences in the turnover rate between polyunsaturated fatty acids of the linoleic acid type and those belonging to the linolenic acid series. Copyright © 1968, Wiley Blackwell. All rights reserved