The composition of vesicles determines the physical state and elasticity of their bilayers. Fatty acid spin labels were incorporated into vesicles, composed of the single chain non-ionic surfactant octaoxyethylenelaurate-ester (PEG-8-L), the sucrose laurate-ester L-595 and cholesterol sulfate (CS) to monitor local dynamic properties of lipid molecules in vesicle bilayers and to study the elasticity of vesicle bilayers. Studies with the spin label probes 5-, 12- and 16-doxyl stearic acid (DSA) indicated that both the order parameter and the rotational correlation times increased when the doxyl group was positioned closer to the headgroup region. These findings indicate that the fluidity of membranes decreased near the headgroup region. Comparing 16-DSA incorporated in vesicle formulations with either 30 or 70 mol% showed no difference in alkyl chain mobility as was reflected by the order parameter. The rotational correlation times, however, showed a slowdown from 0.38 to 0.71 and 1.13 ns when the PEG-8-L molar content was decreased from 100 to 70 and 30 mol% for PEG-8-L:L-595:CS vesicles, respectively. Extrusion measurements indicated an increase in elasticity of vesicle bilayers as the molar content of PEG-8-L was increased from 10 to 90 mol%. Incorporation of cholesterol sulfate stabilizes vesicles and thereby, decreases the elasticity. The increased elasticity correlated excellent with a reduction in the rotational correlation times observed. In conclusion, these results demonstrate that when the molar content of the single chain non-ionic surfactant PEG-8-L in vesicles is increased the elasticity is enhanced and the rotational correlation time is reduced. The enhanced elasticity might contribute to an optimal design of vesicles as drug carriers for transdermal application.