Manoalide, a natural product of sponge, irreversibly inhibits phospholipase A2 (PLA2) by reacting with lysine residues. Cobra venom PLA2 mutants were constructed in which four of the six lysine residues were independently replaced by arginine or methionine, which cannot react with manoalide. The mutants were overexpressed in Escherichia coli, renatured, and purified. The enzyme mutants lacking Lys-6 (K6R and K6M) or Lys-79 (K79R) were inhibited only 40% by manoalide while the native cobra venom PLA2 was inhibited 80% under the same conditions. This means that the manoalide modification of either Lys-6 or Lys-79 accounted for only half of the manoalide inhibition. The double mutant (K6R79R) was not inhibited by manoalide at all. Lys-56 (K56R) and Lys-65 (K65R) mutants were inhibited to the same extent as the native enzyme which indicates that these residues are not responsible for any of the inhibitory effects produced by manoalide. These results demonstrate that the reaction of manoalide with both Lys-6 and Lys-79 can account for all of its inhibition of cobra venom PLA2. The inhibition of PLA2 and its mutants with manoalide did not affect the activity of the enzyme toward monomeric substrate, which suggests that manoalide does not modify the catalytic site residues, that it does not block access to this site, and that its inhibition requires an interface. Furthermore, as with native PLA2, the activation of phosphatidylethanolamine hydrolysis by phosphorylcholine-containing compounds was exhibited by all of the mutants suggesting that none of the lysines examined are essential for this activation.