The short-term effects of vasopressin on free fatty acids and lysophospholipids were investigated in hepatocytes isolated from fed rats. Over the time period 0.25 to 10 min vasopressin decreased the steady-state concentrations of palmitic, stearic and oleic acid measured by gas liquid chromatography in extracts of cells incubated at 0.1 mM extracellular Ca2+. The concentrations of arachidonic and linoleic acids did not change. In hepatocytes labelled with [3H]arachidonic acid and incubated at 1.3 mM extracellular Ca2+ vasopressin or the Ca2+-selective ionophore A23187 increased the rate of accumulation of radioactivity in the incubation medium by 40%. The action of A23187 was dependent on extracellular Ca2+. When hepatocytes labelled with 32Pi were treated with vasopressin, no change in the amounts of [32P]lysophosphatidylethanolamine or [32P]lysophosphatidylcholine was observed. It is concluded that the action of vasopressin on hepatocytes is associated with the release of arachidonic acid or metabolites of arachidonic acid but is not accompanied by a general increase in the steady-state concentrations of free fatty acids and lysophospholipids.