The acidic phospholipase A2 isoform from the spitting cobra Naja mossambica mossambica is activated irreversibly by treatment with a molar equivalent of oleoyl imidazolide. The kinetics of the chemical modification of the enzyme can also be monitored by measuring the large reduction of tryptophan fluorescence, which is accompanied by a distinct red shift. The addition of a single molar equivalent of oleic acid to the enzyme produces an instantaneous reduction in fluorescence but with a barely detectable red shift, confirming that the response to oleoyl imidazolide results from covalent modification of the protein rather than hydrolysis of the reagent. The pH dependence of both activation and fluorescence reduction by oleoyl imidazolide has an optimum rate near pH 8.0. We propose that long-chain fatty acids and long-chain acyl imidazolides bind at a single activation site and that the reaction of the imidazolides involves two protein residues, one of which is a nonessential histidine residue and the other a primary amino group.