Phospholipase A 2 (PLA 2) hydrolyzes phospholipids to free fatty acids and lysolipids and thus initiates the biosynthesis of eicosanoids and platelet-activating factor, potent mediators of inflammation, allergy, apoptosis, and tumorigenesis. The relative contributions of the physical properties of membranes and the structural changes in PLA 2 to the interfacial activation of PLA 2, that is, a strong increase in the lipolytic activity upon binding to the surface of phospholipid membranes or micelles, are not well understood. The present results demonstrate that both binding of PLA 2 to phospholipid bilayers and its activity are facilitated by membrane surface electrostatics. Higher PLA 2 activity toward negatively charged membranes is shown to result from stronger membrane-enzyme electrostatic interactions rather than selective hydrolysis of the acidic lipid. Phospholipid hydrolysis by PLA 2 is followed by preferential removal of the liberated lysolipid and accumulation of the fatty acid in the membrane that may predominantly modulate PLA 2 activity by affecting membrane electrostatics and/or morphology. The previously described induction of a flexible helical structure in PLA 2 during interfacial activation was more pronounced at higher negative charge densities of membranes. These findings identify a reciprocal relationship between the membrane surface properties, strength of membrane binding of PLA 2, membrane-induced structural changes in PLA 2, and the enzyme activation.