Lysophosphatidylcholine (lysoPC) is an arrhythmogenic phospholipid metabolite which accumulates in the ischemic myocardium. Reduced catabolism of lysoPC has been proposed to be one of the biochemical mechanisms responsible for the increase in lysoPC content. In this investigation we compared the microsomal catabolism of exogenous labeled lysoPC in isolated perfused rat and guinea pig hearts. Analysis of the amount of radioactivity in microsomal phosphatidylcholine (PC) and free fatty acid (FFA) was used as an index of the participation in lysoPC clearance by acylation catalyzed by acyl-CoA:lysoPC acyltransferase and deacylation catalyzed by lysophospholipase, respectively. There was no significant difference in the incorporation of radioactivity into rat and guinea pig heart microsomes; however, the patterns of radioactivity in lysoPC metabolites were notably different. Equal participation by deacylation and reacylation was observed in rat microsomes, whereas deacylation was clearly the preferred route for lysoPC clearance in guinea pig microsomes. Modulation of enzyme activity by treatment of the isolated heart with pHMB, a sulfhydryl agent, was used to probe the relationship among acylation, deacylation and the extent of lysoPC clearance. In guinea pig microsomes impairment of lysoPC acylation was not associated with any change in the amount of radioactivity in lysoPC because of a compensatory increase in deacylation. In contrast, impaired deacylation in rat microsomes led to significant elevations in the amount of radioactivity in lysoPC. We conclude, therefore, that in intact perfused rat and guinea pig hearts the relative participation of acylation and deacylation in lysoPC clearance differs. Moreover, we propose that the level of deacylation by lysophospholipase is an important factor in the extent of clearance of lysoPC.