Lysophosphatidylcholine (LPC), an important compound in the immune system, regulates a variety of biological processes. We examined and compared the effect of exogenous LPC on intracellular Ca2+ overload in human Jurkat CD4+ T lymphocytes and mouse CTLL-2 CD8+ T lymphocytes. LPC caused a dose-dependent intracellular Ca2+ level ([Ca2+]i) increase in both Jurkat and CTLL-2 lymphocytes. Pretreatment of cells for 5 min with 30 μM of ruthenium red, a potent ryanodine receptor inhibitor, reduced the LPC-induced Ca2+ response in both Jurkat and CTLL-2 T lymphocytes. Moreover, pretreatment of cells with 100 μM 2-APB for 15 min, a cell-permanent IP3 receptor inhibitor, reduced about two thirds of the LPC induced calcium response in both kinds of cells. However, preincubation of the cells with verapamil, an L-type Ca2+ channel blocker, did not affect the LPC-induced [Ca2+]i increase in CTLL-2 lymphocytes but inhibited this in Jurkat lymphocytes by 26%. In Ca2+-free medium, LPC produced 75.8% of the total [Ca2+]i increase in CTLL-2 lymphocytes and 38% of the total [Ca2+]i increase in Jurkat lymphocytes. These data suggested that the LPC-induced [Ca2+]i increase in human Jurkat and mouse CTLL-2 cell lines occurs via different pathways.