Adenosine 5'-hexadecylphosphate (AMPC16) is a mimic of palmitoyl-AMP, a transient intermediate of the acyl-CoA synthetic reaction, and thus could effectively inhibit the activity of palmitoyl-CoA synthetase of Saccharomyces cerevisiae. AMPC16 inhibited the cellular incorporation of [H-3]palmitic acid into phospholipids, in which such an inhibition was most significantly detected in the fraction containing both phosphatidylinositol (PI) and phosphatidylserine (PS). A pulse-chase experiment revealed the loss of PI and PS accompanied with accumulation of free fatty acids in AMPC16-treated cells. AMPC16-induced events suggested that Faa1p, the major acyl-CoA synthetase isozyme, is functional for phospholipid reconstitution in the exponential growth phase.