Hyperfine coupling constants, differential line widths, and line heights have been determined for the sodium 2,2,5,5-tetramethyl-l-pyrrolidinyloxy-3-carboxylate spin probe, R-.Na+, in the presence of dioctadecyldimethylammonium chloride (DODAC) vesicles by electron paramagnetic resonance (EPR) spectroscopy. Distinct line broadening and asymmetry in the high-field line indicated R-.Na+to be in different environments on the EPR time scale. Line-shape analysis of the EPR spectra, obtained in solutions containing different concentrations of R-.Na+and DODAC, led to a value of 375M-lfor the binding constant between the spin probe and the DODAC vesicle, KR-.Na+. These data also provided information on the amount of water trapped within and associated at the outside of the vesicle. Sodium ascorbate was found to scavenge R-.Na+associated with DODAC vesicles. Rate constants for the scavenging have been determined. Analysis of the kinetics led to a value of KR-.Na+of 328M-1and to a value of 627 M-1for the association constant of sodium ascorbate with the DODAC vesicle. Subsequent to scavenging by sodium ascorbate, a residual concentration of undestroyed R~-Na+ remained in the presence of DODAC. Apparently on the longer time scale, some spin labels enter into the interior of the vesicle where sodium ascorbate cannot reach them. The good agreement of KR-.Na+obtained by the two different methods substantiates the proposed model for single compartment DODAC vesicles. This involves spherical bilayers with an average diameter of 300 A and a vesicle thickness of 50 Å.