Secretory human phospholipase A₂ type IIA (PLA₂-IIA) catalyzes the hydrolysis of the sn-2 ester bond in glycerolipids to produce fatty acids and lysolipids. The enzyme is coupled to the inflammatory response, and its specificity toward anionic membrane interfaces suggests a role as a bactericidal agent. PLA₂-IIA may also target perturbed native cell membranes that expose anionic lipids to the extracellular face. However, anionic lipid contents in native cells appear lower than the threshold levels necessary for activation. By using phosphatidylcholine/phosphatidylglycerol model systems, we show that local enrichment of anionic lipids into fluid domains triggers PLA₂-IIA activity. In addition, the compositional range of enzyme activity is shown to be related to the underlying lipid phase diagram. A comparison is done between PLA₂-IIA and snake venom PLA₂, which in contrast to PLA₂-IIA hydrolyzes both anionic and zwitterionic membranes. In general, this work shows that PLA₂-IIA activation can be accomplished through local enrichment of anionic lipids into domains, indicating a mechanism for PLA₂-IIA to target perturbed native membranes with low global anionic lipid contents. The results also show that the underlying lipid phase diagram, which determines the lipid composition at a local level, can be used to predict PLA₂-IIA activity.