Osmotic pressure (Pi) induces membrane tension in cell membranes and the lipid bilayers of vesicles and plays an important role in the functions and physical properties of these membranes. We recently developed a method to determine quantitatively the membrane tension of giant unilamellar vesicles (GUVs) under. and applied it to GUVs comprising electrically neutral dioleoylphosphatidylcholine (DOPC). Here, we examined the effect of. on GUVs composed of DOPC and negatively charged dioleoylphosphatidylglycerol (DOPG) in a buffer containing a physiological concentration of ions. First, we examined the rate constant, k(r), for constant tension (sigma(ex))-induced rupture of DOPG/DOPC (4/6)-GUVs under Pi and obtained the dependence of k(r) on sigma(ex) in GUVs for various values of Pi. Comparing this dependence in the absence of Pi provided values for membrane tension due to Pi, sigma(osm), which agree with the theoretical values within the experimental error. The values of sigma(osm) for DOPG/DOPC-GUVs were smaller than those for DOPC-GUVs under the same Pi. Two factors, that is, the solute concentration in a GUV suspension and the elastic modulus of the GUV membrane, can reasonably explain this difference based on the theory of sigma(osm). We also examined the effect of Pi on the rate constant, k(FF), for the transbilayer movement of lipid molecules in single GUVs. The values of kFF increased with increasing Pi, indicating that k(FF) increased with sigma(osm). This result supports the existence of prepores in stretched lipid bilayers. Based on these results, we discuss the membrane tension of DOPG/DOPC-GUVs under Pi.