Lysophosphatidylcholine (LPC) is known to increase the intracellular concentration of Ca²⁺ ([Ca²⁺]_i), leading to cell damage. In the present study we examined whether LPC activates phospholipase A₂ (PLA₂) and whether the activation of PLA₂ is responsible for the LPC-induced cell damage in isolated rat cardiomyocytes. LPC (15 µM) produced an increase in [Ca²⁺]_i, a change in cell shape from rod to round, and the release of creatine kinase (CK) accompanied by a significant elevation of the cellular level of nonesterified fatty acids (NEFA), especially arachidonic acid. Three PLA₂ inhibitors, 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid (DEDA), 3-(4-octadecylbenzoyl)acrylic acid (OBAA), and manoalide, attenuated the LPC-induced accumulation of unsaturated NEFA to a similar degree. Nevertheless, whereas both DEDA and OBAA attenuated the LPC-induced increase in [Ca²⁺]_i, change in cell shape, and release of CK, manoalide attenuated none of them. In the Ca²⁺-free solution, LPC did not increase [Ca²⁺]_i with significantly less accumulation of NEFA, but it changed the cell shape from rod to round and increased the release of CK. These results suggest that exogenous LPC increases the PLA₂ activity, which, however, may not be responsible for the LPC-induced damage in cardiomyocytes.