The characteristic "disulfiram syndrome" associated with certain alcohol-drug interactions has been attributed in part to acetaldehydemia and subsequent release of biogenic amines. The ability of pargyline (N-methyl-N-propargylbenzylamine, 1) to cause marked elevations in ethanol-derived blood acetaldehyde (AcH) levels in rodents in a manner similar to that observed with disulfiram is well documented. Unlike disulfiram, however, 1 is not an effective inhibitor of aldehyde dehydrogenase (AlDH) in vitro, suggesting that a metabolite of 1 is the active inhibitory species in vivo. Formation of this inhibitor in vivo appears to be mediated by the hepatic cytochrome P-450 enzymes, as evidenced by the ability of SKF-525A to nullify the pargyline-induced rise in blood AcH after ethanol and by the nearly twofold enhancement of this effect by phenobarbital pretreatment. Indirect evidence from our laboratory suggested that this active pargyline-derived inhibitor of AlDH is propiolaldehyde (HC≡CCHO). We have now succeeded in trapping propiolaldehyde as its semicarbazone by incubating 1 with a phenobarbital-induced rat liver microsomal preparation. Isolation of the semicarbazone, characterization by reference to an authentic sample on TLC, and subsequent release of propiolaldehyde and identification of the latter by its gas chromatographic (GC) retention time and by gas chromatography-mass spectrometry (GC-MS) constitute unequivocal proof that this acetylenic aldehyde is a metabolite of 1. We also present evidence that the propiolaldehyde inhibition of mitochondrial AlDH is irreversible: pretreatment of rat liver mitochondria with propiolaldehyde (1.0 mM) at 38 °C caused time- and temperature-dependent inhibition, and enzymatic activity could not be restored by repeated washing of the mitochondria, suggesting covalent binding to the enzyme.