It is widely accepted that insulin action does not involve inositol phospholipid hydrolysis through the stimulation of a phosphatidylinositol-specific phospholipase C (PI-PLC). This consideration prompted us to investigate the insulin effect on the mechanism leading to the accumulation of diacylglycerol (DAG) and phosphatidic acid (PA) in rat hepatocytes. Basically, insulin induces: (i) a significant increase of both [3H]glycerol and fatty acid labelling of DAG; (ii) a significant increase of PA labelling preceding DAG labelling and paralleled by a decrease of phosphatidylcholine (PC) labelling. These observations, which suggest an insulin-dependent involvement of a phospholipase D, are strengthened by the increase of PC-derived phosphatidylethanol in presence of ethanol. Finally, the observation that the PA levels do not return to basal suggests that other mechanisms different from PC hydrolysis, such as the stimulation of direct synthesis of PA, may be activated.