Ceramide-1-phosphate (C1P), produced by ceramide kinase (CERK), is implicated in the regulation of many biological functions including cell growth and inflammation. C1P is a direct activator of group IVA cytosolic phospholipsase A(2) (PLA2G4A or cPLA(2)α). Although activation of the CERK-C1P pathway causes mitogenic and cytoprotective responses in many cells, the pathway shows cytotoxicity in several cells and the precise mechanism has not been elucidated. In the present study, we examined the effect of human CERK (hCERK) expression on cytotoxicity in two cell lines. Expression of hCERK in CHO cells caused cell rounding and lactate dehydrogenase (LDH) leakage, and co-addition of ceramide enhanced these responses. Expression of hCERK enhanced C1P formation and release of arachidonic acid in Ca(2+) ionophore-stimulated cells. Treatment with 20μM C2-C1P for 24 h caused cell rounding, and the response was significantly decreased by an inhibitor of cPLA(2)α. In L929 cells, expression of hCERK with and without ceramide caused cell rounding and LDH leakage, respectively, and the responses were significantly less in a stable clone of L929 cells lacking cPLA(2)α. These findings suggest the involvement of cPLA(2)α in CERK-C1P pathway-induced cytotoxicity.