The properties of supramolecular aggregates can be strongly influenced by the binding of small polar molecules. Here we describe the use of electron spin resonance to characterize the effect of urea on structural properties of reverse micelles. The electron spin resonance (ESR) spectra of water-soluble charged spin probes, 4-(trimethylammonio)-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (trimethyl tempamine, positively charged) and 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl phosphate (tempo phosphate, negatively charged), as well as the amphiphilic spin label 5-(4,4-dimethyloxazolidin-3-oxyl-2-yl)stearic acid (5-SASL) in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles in isooctane were examined with and without urea at increasing water contents (expressed as Wo, water:surfactant molar ratio). Micellar size, monitored by quasielastic light scattering (QELS), increased with increasing Wo and the increase was steeper in the presence o urea. The spectra of 5-SASL were also sensitive to changes in micellar size. As Wo increased, the spectra changed from those of a probe in the fast motional regime to that of a probe rotating anisotropically in a large particle. The data agree with the results of Haering et al. (J. Phys. Chem. 1988, 92, 3574). Rotational correlation times were calculated for spectra in the fast motion regime (low Wo) yielding particle radii in good agreement with those calculated by QELS. At higher Wo’s, the spectra displayed inner and outer extrema, allowing the calculation of the order parameter, S. The values of S increased with increasing Wo, indicating a decrease in the amplitude of motion of the probe long molecular axis. Making use of the data of Haering et al., the probability of trans conformation along the hydrocarbon chain was also found to increase with Wo. Larger S values and slower motion about the long molecular axis were observed with urea. The isotropic hyperfine coupling constant, a(n), increased with Wo, both with and without urea, indicating that the label is sensitive to the increasing hydration of the head group region. Spectra of the water-soluble probes were also sensitive to changes in Wo and to the addition of urea. At very low Wo (ca. 0.2) the probes yielded spectra indicative of orientation, both molecules rotating preferentially about the nitroxide x axis. As the water content increased, the probes displayed different spectral behavior : while tempo phosphate yielded lineshapes and an values similar to those observed in bulk aqueous medium, the spectra of trimethyl tempamine indicated that the molecule was oriented, rotating preferentially about the nitroxide x axis at all Wo’s examined, suggesting that the probe remained at the water-amphiphile interface region. Urea does not displace the probe from this region. Nevertheless, as suggested by the effects on line shapes and a(n), the additive binds at the interface. In agreement with the results obtained for 5-SASL, the spectra of trimethyl tempamine were suggestive of slower probe motion in the presence of urea.