The recent discovery of neutral active choline and ethanolamine glycerophospholipid specific phospholipase C in myocardium (Wolf RA, Gross RW. J Biol Chem 1985;260:7295) has demonstrated a novel catabolic pathway that potentially contributes to the accumulation of amphiphilic metabolites during myocardial ischemia. To assess the potential importance of this pathway, we quantified the temporal course of alterations in myocardial 1-0-alk-1'-enyl-2-acyl-sn-glycerol (AAG) and 1,2-diacyl-sn-glycerol (DAG) content during control and ischemic intervals in an isolated perfused Langendorf model. AAG accumulated over 5-fold to 8.70 and 18.27 nmol/g dry in 20- and 60-minute ischemic rabbit hearts, respectively (p<0.02). The only AAG molecular species that was detected in substantial amounts in control or ischemic rabbit hearts was 1-0-hexadec-1'-enyl-2-acyl-sn-glycerol. Since this molecular species is enriched in plasmenylcholine these findings suggest that AAG production is likely mediated by phospholipase C-catalyzed hydrolysis of plasmenylcholine. In contrast to ischemia-induced AAG accumulation, DAG content decreased during both control and globally ischemic perfusion intervals. In summary, these findings demonstrate that AAG, in contrast to DAG, accumulates during myocardial ischemia indicating that at least some metabolites of plasmalogen and diacyl phospholipids accumulate at differential rates during myocardial ischemia.