We have developed an assay for studying myocardial phospholipase A2 activity by measuring accumulation of lysophospholipids resulting from hydrolysis of the endogenous choline glycerophospholipid pool. This assay was used to characterize phospholipase A2 activity in rabbit myocardium. Lyophilized rabbit myocardium was incubated at 37 degrees C in Tris-HCl buffer containing either ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA)/EDTA or calcium, and palmitoyl-lysophosphatidylcholine (P-LPC), oleoyl-LPC, stearoyl-LPC, and 16:0-lysoplasmenylcholine were measured using a recently developed HPLC method. The identity of the individual species was confirmed by ion-spray LC-MS-MS. In the presence of EGTA/EDTA, incubation for up to 30 min caused a linear increase in all lysophospholipids. The main increases were found in P-LPC and 16:0-lysoplasmenylcholine, which increased by 37 +/- 3 (mean +/- SE, N = 8) and 48 +/- 3 nmol/g dry wt x min, respectively. The apparent phospholipase A2 activity was found to be calcium, temperature, and pH sensitive. The pH optimum was between 6.5 and 8.0, and incubation at room temperature and 45 degrees C decreased the activity by 80 and 40%, respectively. Studies of the metabolism of the formed lysophospholipids showed a substantial metabolism of the lysophospholipids that accounted for about 40% of the total phospholipase A2 activity. This method offers a novel approach to study phospholipase A2 activities by measuring accumulation of products resulting from hydrolysis of endogenous phospholipid pools.