The purified phospholipase A 2 of Naja nigricollis venom is a basic, relatively toxic protein, while the purified phospholipase A 2 of Hemachatus haemachatus is neutral and relatively non-toxic. In order to establish whether the difference in toxicity correlates with hydrolytic ability, we compared the two enzymes using substrates in various physical states such as mixed micelles, native soluble lipoprotein or organized in membranes. The purified phospholipids were used as mixed micelles with Triton X-100. When compared on purified egg l-α-phosphatidylcholine (PC), the two enzymes showed similar pH-and temperature-dependence and were equally affected by activators and inhibitors. N. nigricollis phospholipase A 2 had a V max of 250 μ-equiv. per min per mg and a K m of 4.2 mM, while H. haemachatus phospholipase A 2 had a V max of 1052 μ-equiv. per min per mg and a K m of 2.2 mM. Both enzymes favored the substrates in the liquid-crystalline state. With a buffered egg yolk dilution as substrate, a V max of 356 μ-equiv. per min per mg and a K m of 29 mM were found for N. nigricollis, while H. haemachatus had a V max of 616 μ-equiv. per min per mg and a K m of 25 mM. The hydrolysis of purified PC, l-α-phosphatidylethanolamine (PE), l-α-phosphatidylserine (PS) and l-α-phosphatidylinositol (PI) was followed with the substrates taken either singly or in various combinations. Significant differences in preference of the two enzymes were apparent on single substrates, such as the comparatively high hydrolysis of PC by H. haemachatus phospholipase A 2 and of PE by N. nigricollis phospholipase A 2. On mixtures of the four substrates, taken either in equal amounts or in proportions resembling the phospholipid distribution of human red cells, rat brain or electric eel Sachs organ, the sequence of substrate preference exhibited by the two enzymes was again strikingly different. A main feature of the N. nigricollis phospholipase A 2 was its high ability to hydrolyze PS. There was no essential difference between the actions of the two enzymes on fresh human red cells. However, erythrocytes from stored, outdated blood were hemolyzed and phospholipids were fully hydrolyzed by N. nigricollis phospholipase A 2, while the H. haemachatus enzyme was nonhemolytic and induced only limited hydrolysis. The same disparity in behavior could be demonstrated on fresh guinea pig erythrocytes. A comparison of hydrolysis, in permeable red cell ghosts and in Triton-solubilized membranes, by the phospholipases, revealed that the high preference of N. nigricollis enzyme for PS was masked by sequestration of this phospholipid within the ghosts.