We report here the discovery of a new class of phospholipases A2 in which Asp-49, a residue considered to be an obligate component of the catalytic apparatus, is replaced by a lysine. Asp-49 is invariant among the more than 30 venom and pancreatic phospholipases A2 sequenced to date, and its beta-carboxylate group has been shown to be a ligand for calcium in a binding site which also involves contributions from the peptide carbonyl oxygens of Tyr-28, Gly-30, and Gly-32, the so-called calcium-binding loop. The change of Asp-49 to a lysine, and other substitutions in regions heretofore thought to be invariant, including the calcium-binding loop, suggested that the new phospholipases might differ functionally with respect to calcium and/or substrate binding. Indeed, although the Lys-49 phospholipases A2 show a dependence on calcium similar to that of the Asp-49 enzymes, they may be distinguished by the fact that, in the absence of phospholipid, they do not bind calcium to any measurable extent under conditions where Asp-49 enzymes bind a stoichiometric amount of calcium. Furthermore, in the absence of calcium, they show binding to single bilayer phospholipid vesicles under conditions where Asp-49 phospholipases do not bind at all. These results suggest a reversed order of addition of calcium and substrate in the formation of the ternary catalytic complex in the Lys-49 phospholipases A2. Although the mechanistic implications of these structural and functional alterations are not defined at present, it is clear that Asp-49 is not essential for phospholipase A2 catalysis and that it does not participate in the enzyme-calcium-phospholipid catalytic complex.