Phosphatidylalcohols, such as phosphatidylethanol (PEth), are formed from phosphatidylcholine in the presence of a primary alcohol (e.g., ethanol). This 'transphosphatidylation' reaction is used as specific phospholipase D (PLD) assay. Accumulation of PEth in tissues is recognized as a reliable measure of PLD activity, as PEth is allegedly metabolically stable. The general validity of this assumption was reinvestigated in isolated rat heart, small intestine and brain slices. The half-times of 3H-PEth degradation (labelled with 3H-myristic acid and preformed by ethanol exposure for 30 min) were about 1 h in heart and small intestine, but 17 h in brain. As the formation of PEth is superimposed by simultaneous degradation, a mathematical model was established to calculate the differences between 'true' and 'apparent' PEth formation. As expected, this difference was relevant in heart and intestine, but not in brain tissue. For example, ischemia in the perfused heart for 30 min reversibly blocked PEth degradation and seemingly enhanced PEth formation; the block was reversed by ischemic preconditioning (IPC) and by pretreatment with diazoxide, an opener of mitochondrial K(ATP) channels. In conclusion, PEth degradation in heart was energy-dependent and rapid, which, when ignored, may lead to misinterpretation of PEth values with respect to PLD activity.