The effect of diethylstilbestrol injection on the activities of phosphatidylcholine biosynthetic enzymes in rooster liver has been determined. Choline kinase activity was stimulated within 4 h after the first hormone injection. By the third day enzyme activity reached 5.47 nmol . min-1 . mg-1 protein compared to control values (1.83 nmol . min-1 . mg-1 protein) which were unchanged during the the experiment. CTP : phosphocholine cytidylyltransferase activity was unaffected until Day 3 when its activity was 50% that of control values. When assayed in the presence of exogenous phospholipid, no significant change was noted in cytidylyltransferase activity. The activity of CDPcholine : 1,2-diacylglycerol phosphocholinetransferase was not altered by the hormone injections. The activity of phosphatidylethanolamine-N-methyltransferase gradually increased so that by Day 3, the enzyme activity was elevated 2-fold (0.12 to 0.24 nmol methyl group transferred per mg microsomal protein). These results are consistent with earlier in vivo studies (Vigo, C. and Vance, D.E. (1981) Eur. J. Biochem., in the press) that indicated a stimulation of phosphatidylcholine biosynthesis via CDPcholine during the first 2 days of diethylstilbestrol injection and inhibition on the third day.