Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of group IV cytosolic phospholipase A(2)alpha (cPLA(2)alpha) via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic beta-groove (Arg(57), Lys(58), Arg(59)) of the C2 domain of cPLA(2)alpha. In this regard, mutants of this region of cPLA(2)alpha were generated (R57A/K58A/R59A, R57A/R59A, K58A/R59A, R57A/K58A, R57A, K58A, and R59A) and examined for C1P affinity by surface plasmon resonance. The triple mutants (R57A/K58A/R59A), the double mutants (R57A/R59A, K58A/R59A, and R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P-containing vesicles as compared with wild-type cPLA(2)alpha. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA(2)alpha also showed a significant reduction in the ability of C1P to: 1) increase the V(max) of the reaction; and 2) significantly decrease the dissociation constant (K (A)(s)) of the reaction as compared with the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA(2)alpha demonstrated normal basal activity as well as normal affinities for phosphatidylcholine and phosphatidylinositol-4,5-bisphosphate as compared with wild-type cPLA(2)alpha. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic beta-groove of the C2 domain of cPLA(2)alpha.