The activity of the major isoform of porcine pancreatic phospholipase A2 (PLA2), designated B-PLA2, against micellar substrates is inhibited by heparin. Inhibition is a consequence of binding of the enzyme to heparin, documented by a heparin-induced alteration in the intrinsic fluorescence of B-PLA2 and in the 8-anilino-1-naphthalene sulfonate fluorescence and by the enhanced rate of chemical modification of the active site residue His-48. As a consequence of heparin binding, the conformation of B-PLA2 at the active site and at the amino-terminus is altered, and the enzyme does not bind to phospholipid micelles. In spite of the heparin-induced conformational changes, B-PLA2 retains its ability to catalyze the hydrolysis of monomeric phospholipid. Other glycosaminoglycans can bind to and inhibit the activity of B-PLA2 toward organized phospholipids, but none tested is as effective as heparin. An isoform of the pancreatic enzyme, designated UB-PLA2 and which corresponds to iso-pig PLA2, does not bind to nor is its catalytic activity influenced by heparin. A peptide corresponding to the amino-terminal 26 residues of B-PLA2 can rescue PLA2 from heparin inhibition. A similar peptide corresponding to the amino-terminus of UB-PLA2 has no effect on heparin inhibition. A model for the inhibition of B-PLA2 by heparin is proposed in which the catalytically significant effect of heparin is to interact directly with the amino-terminus of B-PLA2, the interfacial recognition site, to prevent the enzyme from binding to micellar substrates.