Although it is known that the antioxidant properties of melatonin can be modulated by its effect on membrane fluidity, there are few studies on this subject reported in the literature and they are controversial. In this study, viscosimetry and nuclear magnetic resonance (NMR) techniques were used to determine melatonin's effect and location on egg phosphatidylcholine bilayers mobility. Melatonin decreases the dynamic viscosity of the lipid dispersion. (31)P-NMR line width analysis indicated that melatonin induces a slight but uniform restriction of the lipid motional freedom in the polar head. However, melatonin changes in choline (13)C dynamics was only observed through chemical shift analysis. On the other hand, melatonin can induce an increase in the lipid nonpolar chain mobility, as observed by (13)C and (1)H relaxation time analysis. These results suggest the interfacial location of melatonin in the membrane. Additionally, the results of the analysis of the lipid (1)H-fitted exponential relaxation times suggest that melatonin promotes a molecular rearrangement of the bilayers. The melatonin effect and location in the lipid membrane may explain its antioxidant properties against lipid peroxidation induced by reactive species.