A rapid and accurate method has been developed for measuring the elastic response of vesicle bilayer membranes to an applied osmotic pressure. The technique of dynamic light scattering is used to measure both the elastic constant and the elastic limit of dioleoylphosphatidic acid (DOPA) and DOPA-cholesterol vesicles and of submitochondrial particles derived from the inner membrane of bovine heart mitochondria. The vesicles prepared by the pH-adjustment method are unilamellar and of uniform size between 240 and 460 nm in diameter. The vesicles swell uniformly upon dilution. The observed change in size is not due to any change in the shape of the vesicles. The data also indicate that the vesicles are spherical and not flaccid. The total vesicle swelling in these studies resulted in a 3-4% increase in surface area for vesicles swollen in 0.15 M KCl and a 5-10% increase in surface area for vesicles swollen in 0.25 M sucrose. This maximum represents the elastic limit of the vesicles. Evidence is presented to show that the vesicles release contents after swelling to this maximum, reseal immediately, and reswell according to the osmotic pressure. For DOPA vesicles in a 0.15 M KCl-tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer (pH 7.55), the observed membrane modulus is found to be in the range of 10(8) dyn/cm2. The modulus was found to be in the order of 10(7) dyn/cm2 for DOPA vesicles in a 0.25 M sucrose-Tris-HCl buffer (pH 7.55). This is comparable to that of submitochondrial particles in the same sucrose-Tris-HCl buffer. The observed membrane modulus also decreases with vesicle size. Its magnitude and its variation with ionic strength indicate that the major component of bilayer elasticity is neither the inherent elasticity of the bilayer nor the bending modulus. The variation of the membrane modulus with respect to curvature suggests that its principal component may be related to surface tension effects including the negative charges on the vesicle surface. There is considerable variation between vesicles swollen in sucrose and those swollen in KCl in the membrane modulus, in the elastic limit at which the vesicles burst, and in the transbilayer pressure difference at bursting. The latter was found to be 4-6 mosM (10(5) dyn/cm2) in sucrose solution and 20-4 mosM (10(6) dyn/cm2) in KCl solution.