Phospholipase A2 (Naja naja naja) catalyzes the hydrolysis of dipalmitoyl phosphatidylcholine in small unilamellar vesicles (SUVs) with a faster initial rate than in large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). For the SUVs, the hydrolysis was initially faster for gel phase than liquid crystalline phase phospholipid. For both LUVs and MLVs, hydrolysis was low except in a small temperature range around the thermotropic phase transition of the phospholipid. In this temperature range, the reaction time course of phospholipase action on dipalmitoyl phosphatidylcholine in LUVs and MLVs included a lag period. With SUVs, a lag period also was observed above the phase transition temperature, but it was not observed below it.