When 388D1 cells are incubated in media containing 10 microM [3H]arachidonic acid (delta4Ach), the label is rapidly incorporated into phospholipids and triacylglycerol. However, incorporation of [3H]delta4Ach into phospholipids clearly precedes incorporation into triacylglycerol, indicating that the phospholipid pool constitutes the primary metabolic fate of the delta4Ach via a remodelling pathway. In contrast, [3H]delta4Ach is incorporated into triacylglycerol almost exclusively via de novo synthesis, as evidenced by studies using propranolol, a phosphatidate phosphohydrolase inhibitor. This compound induced a time-dependent and concentration-dependent increase in the levels of [3H]delta4Ach-containing phosphatidate that is directly correlated with a decrease in the levels of [3H]delta4Ach-containing triacylglycerol. In addition, no change in the levels of [3H]delta4Ach-containing diacylglycerol and monoacylglycerol were apparent along the time course of fatty acid incorporation into triacylglycerol. However, a sharp and transient accumulation of cell-associated free [3H]delta4Ach was detected shortly after exposure of the cells to the radioactive fatty acid. Collectively, the results reported herein suggest that free delta4Ach availability determines the patterns of incorporation and distribution of this fatty acid among the various lipid classes of P338D1 cells.