The action of the phospholipases A2 (PLA2s) from Naja naja naja, Naja naja atra, and Crotalus atrox venoms as well as the enzyme from porcine pancreas on a number of short-chain, water-soluble substrates was studied. The inhibition of these enzymes by short-chain phosphonate- and thiophosphonate-containing phospholipid analogues was also examined. The kinetic patterns observed for the action of the venom PLA2s on substrates containing phosphocholine head groups all deviated from a classical Michaelis-Menten-type behavior. With a substrate containing an anionic head group, the kinetic pattern observed was more normal. In contrast, Michaelis-Menten-type behavior was observed for the action of the porcine pancreatic PLA2 acting on all of the substrates studied. A short-chain phospholipid analogue in which the enzyme-susceptible ester was replaced with a phosphonate group was found to be a tight-binding inhibitor of the venom PLA2s with IC50 values that were some 10(4)-10(5)-fold lower than the concentration of substrate used in the assay. The degree of inhibition was found to depend dramatically on the stereochemical arrangement of substituents in the inhibitor which strongly suggests that the inhibitors are binding directly to the active site of the PLA2s. By comparison, the phosphonate analogue functioned as a poor inhibitor of the porcine pancreatic PLA2. Direct inhibitor binding studies indicated that the short-chain phosphonate inhibitor bound weakly to the venom enzymes in the absence of the short-chain substrates. Several other unusual features of the inhibition were also observed. The data are interpreted in terms of a model in which the enzyme and substrate form a lipid-protein aggregate at substrate concentrations below the critical micelle concentration (cmc). Possible reasons for the selective binding of the inhibitor to the enzyme-substrate microaggregate are discussed.