The addition of 1 microM-vasopressin or -angiotensin to isolated rat hepatocytes induced a fast transient inhibition of the rate of incorporation of [Me-3H]choline into phosphatidylcholine. The cationophore A23187 induced a similar inhibition of phosphatidylcholine synthesis. The addition of micromolar Ca2+ to rat liver microsomes inhibited the activity of CDP-choline: 1,2-diacylglycerol cholinephosphotransferase. This inhibition is due a decrease in the Vmax. of the enzyme without affecting the Km for CDP-choline. It is concluded that Ca2+ regulates phosphatidylcholine synthesis in rat liver.