Extracellular 14-kDa phospholipases A2 (PLA2) in inflammatory exudates can contribute to bacterial phospholipid (PL) degradation during phagocytosis of Escherichia coli by polymorphonuclear leukocytes (PMN) and are highly active toward E. coli treated with the bactericidal/permeability-increasing protein (BPI) purified from PMN. PLA2 activity toward BPI-treated E. coli varies greatly among members of this conserved family of enzymes and apparently depends on a cluster of basic residues in a variable surface region near the NH2 terminus for recognition of this biological target (Weiss, J., Wright, G.W., Bekkers, A.C.A.P.A., van den Bergh, C.J., and Verheij, H.M. (1991) J. Biol. Chem. 266, 4162-4167). We have examined by site-specific mutagenesis of a recombinant PLA2 that is identical to an enzyme in human synovial fluid (containing His-6, Arg-7, Lys-10, and Lys-15 and a global net charge of +15) the role of basic residues in this region in PLA2 action against PLA-deficient (pldA-) E. coli. Substitution of Ser for Arg-7 +/- Gln for Lys-15 caused, respectively, about a 10- and 25-fold reduction in BPI-dependent PLA2 binding and activity to E. coli, but had no effect on hydrolysis of PL of autoclaved E. coli or dispersions of purified PL. PL degradation during phagocytosis was increased after pretreatment of E. coli (or PMN) with wild-type PLA2 followed by removal of unbound PLA2. Thus, the PLA2 binds to cells before phagocytosis followed by internalization of the enzyme along with E. coli and intracellular action. Mutant (e.g. R7S +/- K15Q) PLA2 show the same BPI-independent binding to E. coli as the wild-type enzyme but 10-30-fold reduced activity during phagocytosis, reflecting lower intracellular activity of these enzymes. Thus, structural determinants first implicated in PLA2 action toward E. coli treated with purified BPI apparently are also important in the intracellular action of PLA2 during phagocytosis by PMN.