Polymyxin B (Px), a cyclic cationic peptide, was shown to act as a potent activator of interfacial catalysis by phospholipase A2 (PLA2) acting on dimyristoylphosphatidylmethanol vesicles in the scooting mode. A 7-fold increase in the initial enzymatic velocity was seen with the pig pancreatic PLA2 in the presence of 1 microM Px. Initial experiments including the dependency of the degree of activation by Px on the source of the PLA2 suggested that Px bound to a cationic binding site on the enzyme. However, numerous additional observations led to the conclusion that activation by Px was due to its effects on the substrate interface. For example, the activation by Px was only seen when the PLA2 acted on small vesicles rather than larger ones, and all of the available substrate was eventually hydrolyzed in the presence of a small mole fraction of Px. Px did not promote the intervesicle exchange of PLA2, and it did not alter the binding of the evidence led to the conclusion that Px activated interfacial catalysis by promoting the replenishment of substrate in the enzyme-containing vesicles. When PLA2 was acting on small vesicles in the scooting mode, the observed initial velocity was lower than that measured with large vesicles because the surface concentration of substrate decreased relatively rapidly in the small vesicles. Px promoted the transfer of phospholipids between the vesicles and functioned as an activator by keeping the mole fraction of substrate in the enzyme-containing vesicles close to 1. This effect of Px was consistent with the ability of polycationic peptides to induce the intervesicle mixing of anionic phospholipids in vesicles [Bondeson, J., & Sundler, R. (1990) Biochim. Biophys. Act 1026, 186-194]. Activation by substrate replenishment was quantitatively predicted by the theory of interfacial catalysis on vesicles in the scooting mode. The role of substrate replenishment in the kinetics of interfacial catalysis in phospholipid micelles was discussed. Finally, the protocols developed in this paper were outlined in view of their utility in the analysis of activators of interfacial catalysis.