PEG-12-acyloxystearates constitute a novel class of pharmaceutical solubilizers and are synthesized from polyethylene glycol and 12-hydroxystearic acid, which has been esterified with a second acyl chain. The hemolytic activity of these surfactants decreases drastically with increasing pendant acyloxy chain length, and surfactants with an acyloxy chain of 14 carbon atoms or more are essentially nonhemolytic. In this paper, the interactions of PEG-12-acyloxystearates (acyloxy chain lengths ranging from 8 to 16 carbon atoms) with phosphatidylcholine vesicles, used as a model system for erythrocyte membranes, were studied in search of an explanation for the large variations in hemolytic activity. Surfactant-induced alterations of membrane permeability were investigated by studying the leakage of vesicle-entrapped calcein. It was found that all of the surfactants within the series interact with the vesicle membranes and cause slow leakage at elevated surfactant concentrations, but with large variations in leakage kinetics. The initial leakage rate decreases rapidly with increasing pendant acyloxy chain length. After prolonged incubation, on the other hand, the leakage is not a simple function of acyloxy chain length. The effect of the surfactants on membrane integrity was also investigated by turbidity measurements and cryo-transmission electron microscopy. At a surfactant/lipid molar ratio of 0.4, the vesicle membranes are saturated with surfactant. When the surfactant/lipid molar ratio is further increased, the vesicle membranes are progressively solubilized into mixed micelles. The rate of this process decreases strongly with increasing acyloxy chain length. When comparing the results of the different experiments, it can be concluded that there is no membrane permeabilization below saturation of the vesicle membranes. The large variations in the kinetics suggest that several steps are involved in the mechanism of leakage induced by PEG-12-acyloxystearates and that their relative rates vary with acyloxy chain length. The slow kinetics may in part be explained by the low critical micelle concentrations (CMCs) exhibited by the surfactants. The CMCs were found to be in the range of 0.003-0.025 microM.