The effects of vasopressin on the short-term control of fatty acid metabolism were studied in isolated rat hepatocytes. Vasopressin increased the oxidation of oleate to CO2 and decreased the formation of ketones in hepatocytes from Wistar rats, but not from Brattleboro rats. Incubation with vasopressin for 30 min increased the conversion of oleate into triacylglycerol by 17% and 32% in hepatocytes from Wistar and Brattleboro rats respectively. The corresponding increases for the phospholipid fraction were 19% and 42%. When Wistar-rat hepatocytes were incubated with corticosterone for 6 h there was a 19% increase in triacylglycerol synthesis, and a 52% increase if vasopressin was added 30 min before the end of the incubation. Glycerol phosphate acyltransferase activity was not significantly increased by vasopressin. Incubation for 5-60 min with vasopressin increased the Vmax. of phosphatidate phosphohydrolase by 48% and 32% respectively in hepatocytes from Wistar and Brattleboro rats. These increases were antagonized if EGTA was added to the medium used for incubating the hepatocytes. The replacement of vasopressin by 5 microM-ionophore A23187 produced a significant increase of 13% in the phosphohydrolase activity. It is therefore likely that the effects of vasopressin on the phosphohydrolase are mediated by Ca2+. These results are discussed in relation to the possible function of phosphatidate phosphohydrolase in controlling the turnover of phosphoinositides, the synthesis of phosphatidylethanolamine, phosphatidylcholine and triacylglycerol, and the secretion of very-low-density lipoproteins.