A long-chain acyl-CoA synthetase (Faap) inhibitor, adenosine 5'-hexadecylphosphate (AMPC16), caused lethal plasma membrane damage to Saccharomyces cerevisiae cells as reflected by the leakage of cytoplasmic K+ into medium in which Mg2+ was supplemented at 10 mM. AMPC16 did not interfere with the de novo synthesis of phospholipids using acetyl-CoA as a starting material, but the AMP analog accelerated the liberation of long- chain fatty acids from phospholipids with the aid of exogenous Mg2+, suggesting that the role of this divalent cation is to maximize the rate of the acyl group turnover. This Mg2+-dependent fungicidal effect of AMPC16 was similarly observed with a mutant lacking any of the phospbolipase B (Plbp) isozymes, whereas it was mostly suppressed in a phospholipase C (Plc1p) deletion mutant in which the liberation of fatty acids from phospholipids was completely prevented. These results suggest that Plc1p is normally functional for phospholipid reconstitution in exponentially growing cells and enhancement of its activity by exogenous Mg2+ could be a cause of the irreversible deacylation of plasma membrane phospholipids when their reacylation is blocked at the step of activation of long-chain fatty acids by one of the Faap isozymes.