Previous findings from our laboratory have demonstrated that cPLA(2)alpha is directly activated by the emerging bioactive sphingolipid, ceramide 1-phosphate (C-1-P) (1). In this study, a Triton X-100/phosphatidylcholine (PC) mixed micelle assay was utilized to determine the kinetics and specificity of this lipid-enzyme interaction. Using this assay, the addition of C-1-P induced a dramatic increase in the activity of cPLA(2)alpha (>15-fold) with a K(a) of 2.4 mol % C-1-P/Triton X-100 micelle. This activation was highly specific as the addition of other lipids had insignificant effects on cPLA(2)alpha activity. Studies using surface-dilution kinetics revealed that C-1-P had no effect on the Michaelis-Menten constant, K(m)(B), but decreased the dissociation constant (K (A)(s)) value by 87%. Thus, C-1-P not only increases the membrane affinity of cPLA(2)alpha but also may act as an allosteric activator of the enzyme. Surface plasmon resonance analysis of the C-1-P/cPLA(2)alpha interaction verified a decrease in the dissociation constant, demonstrating that cPLA(2)alpha bound PC vesicles containing C-1-P with increased affinity (5-fold) compared with PC vesicles alone. The effect on the dissociation rate of cPLA(2)alpha was also found to be lipid-specific with the exception of phosphatidylinositol 4,5-bisphosphate, which caused a modest increase in vesicle affinity (2-fold). Lastly, the binding site for C-1-P was determined to be within the C2-domain of cPLA(2)alpha, unlike phosphatidylinositol 4,5-bisphosphate. These data demonstrate a novel interaction site for C-1-P and suggest that C-1-P may function to recruit cPLA(2)alpha to intracellular membranes as well as allosterically activate the membrane-associated enzyme.