The effects of phospholipid vesicles and divalent cations in the subphase solution on the surface tension of phospholipid monolayer membranes were studied in order to elucidate the nature of the divalent cation-induced vesicle membrane interaction. The monolayers were formed at the air/water interface. Various concentrations of unilamellar phospholipid (phosphatidylserine, phosphatidylcholine and their mixtures) vesicles and divalent cations (Mg2+, Ca2+, Mn2+, etc.) were introduced into the subphase solution of the monolayers. The changes of surface tension of monolayers were measured by the Wilhelmy plate (Teflon) method with respect to divalent ion concentrations and time. When a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1 : 1) were used, there were critical concentrations of divalent cations to produce a large reduction in surface tension of the monolayer. These concentrations were 16 mM for Mg2+, 7 mM for Sr2+, 6 mM for Ca2+, 3.5 mM for Ba2+ and 1.8 mM for Mn2+. On the other hand, for a phosphatidylcholine monolayer and phosphatidylcholine vesicles, there was no change in surface tension of the monolayer up to 25 mM of any divalent ion used. When a phosphatidylserine monolayer and phosphatidylcholine vesicles were used, the order of divalent ions to effect the large reduction of surface tension was Mn2+ greater than Ca2+ greater than Mg2+ and their critical concentrations were in upon vesicle concentrations as well as the area/molecule of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine : 1) vesicles, above the critical concentrations of Mn2+ and Ca2+, the surface tension decreased to a value close to the equilibrium pressure of the monolayers within 0.5 h. This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes. The order and magnitude of divalent cation concentrations at which phosphatidylserine/phosphatidylcholine (1 : 1) and phosphatidylserine vesicle suspensions showed a large increase in turbidity were similar to those obtained in the above mentioned experiments.