Human nonpancreatic (group IIa) secreted phospholipase A2 (human sPLA2) is associated with a number of inflammatory disorders in which the extracellular concentrations of this enzyme can become highly elevated. It is probable that the enzyme normally acts as an acute-phase protein whose function is to facilitate the removal of infectious organisms or damaged host cells as part of the normal inflammatory response. The enzyme shows negligible activity with phosphatidylcholine (PC) vesicles and cell membranes, presumably reflecting the enzyme's lack of ability to bind productively to such condensed neutral interfaces. Mammalian pancreatic enzymes show modest activity with such interfaces and contain a unique tryptophan at position 3, which is part of the presumptive interfacial binding surface of these enzymes. Human sPLA2 does not contain tryptophan. The amphiphilic indole side chain of tryptophan is noted for its ability to penetrate the lipid interface of membranes, and tryptophan residues appear to be associated with the ability of lipases and phospholipases A2 to bind to and hydrolyze such interfaces. We have investigated in detail the properties of a V3W mutant of human sPLA2, which has a unique tryptophan on the interfacial binding surface of this enzyme. Although this enzyme shows a modest ( approximately 50%) reduction in activity when anionic substrates are used under standard assay conditions, the activity of the enzyme on phosphatidylcholine vesicles and cell membranes is dramatically increased compared with human sPLA2. This is particularly the case with small unilamellar vesicles of PC, where activity is enhanced over 250-fold compared to the almost zero activity expressed by human sPLA2. This enhanced activity is best explained by increased interfacial binding and activation of the V3W mutant and is not due to enhanced active-site binding and hydrolysis. The results highlight the important role that tryptophan residues can play in interfacial binding, particularly to condensed zwitterionic interfaces. The interfacial characteristics of the mutant human enzyme now resemble more closely the mammalian pancreatic enzymes that already have a tryptophan at position 3.