Clones of HepG2 cells were selected that stably express the cDNA for hormone-sensitive lipase (HSL). When cells were cultured in the presence of labelled extracellular oleate, accumulation of labelled fatty acid as cellular triacylglycerol (TAG) was significantly lower in the transfectants compared with the wild-type cells. There was no change in the net rate of phospholipid (PL) synthesis. Culture of cells containing isotopically prelabelled TAG resulted in a greater net loss of TAG from the transfected cells than from the wild-type cells. The excess loss of labelled TAG was primarily due to an increased TAG fatty acid oxidation. Free fatty acid release into the medium was not increased in the transfectants, nor was the very low rate of lipoprotein lipid secretion. Also, there was no increased net trafficking of fatty acids from TAG into PLs. Changes in the 3H:14C ratio of TAG prelabelled with [3H]glycerol and [14C]oleate suggested that none of excess TAG fatty acid released in the transfected cells underwent intracellular re-esterification to TAG prior to oxidation. The results suggest that fatty acids mobilized by HSL are directed immediately into the oxidative pathway and are not available for biosynthetic processes. It appears likely, therefore, that intracellular TAG-derived fatty acids which enter the oxidative pathway exist in a different compartment from those that are directed towards synthesis.