The effect of vitamin B_6 deficiency on the biosynthesis of arachidonic acid (20 : 4n-6) from linoleic acid (18 : 2n-6) in rat liver microsomes was studied. Rats fed on a vitamin B_6-deficient diet containing 70% casein for 5 weeks displayed a striking decrease mainly in the activity of terminal Δ6-desaturase (D6D) of the 18 : 2n-6 desaturation system. The decreased activity of D6D seemed to be due to a decrease in the microsomal content of phosphatidylcholine (PC). The activities of choline phosphokinase and choline phosphotransferase decreased clearly. Additionally, the molar ratio of S-adenosylmethionine/S-adenosylhomocysteine in liver decreased drastically and the methylation of phosphatidylethanolamine to PC was repressed. Results in this study demonstrate that vitamin B_6 deficiency modified methionine metabolism and decreased f\choline utilization, which indirectly affected PC biosynthesis, and finally depressed the 18 : 2n-6 desaturation which brought about the decrease of the biosynthesis of 20 : 4n-6.