Fusion of phospholipid vesicles composed of various combination of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid was induced by Ca2+ in the presence or absence of phospholipase D from Streptomyces chromofuscus. For the vesicles composed of 20:50:30 molar ratio of phosphatidylcholine/phosphatidylethanolamine/phosphatidic acid, the initial fusion rate was much faster than the expected value when only the conversion of phosphatidylcholine and phosphatidylethanolamine into phosphatidic acid by phospholipase D is taken into account. These observations are discussed in terms of the involvement of outer monolayer of the vesicles and the enzyme activity itself in the fusion process.