We studied the formation and stability of vesicles consisting of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and phosphatidylcholines by electron spin resonance (ESR) analysis and observation of their hemolytic activities. In contrast with previous findings on dimethyldialkylammoniums, DOTAP formed vesicles at 37 degrees C with phosphatidylcholines containing either saturated acyl chains such as dimyristoylphosphatidylcholine (DMPC) or unsaturated acyl chains such as dilinoleoylphosphatidylcholine (DLPC). Phosphatidylcholines made the bilayer more rigid and significantly reduced the hemolytic activity of DOTAP. In the presence of equimolar concentration of DOTAP and phosphatidylcholines, formation of tightly aggregated structures of several erythrocytes was observed, as previously reported for the vesicles containing dimethyldipalmitylammonium. These findings indicate that DOTAP vesicles were stabilized by phosphatidylcholines with either saturated acyl chains or unsaturated acyl chains, and the interaction with the lipid bilayer of biological membranes as cationic vesicles became prominent with minimal membrane damage by DOTAP monomers.