The physical properties of lipids can have a major influence on lipid oxidation reactions. Edible oils contain surface-active compounds and water that can form physical structures such as reverse micelles. This study used the fluorescence probe, 5-dodecanoylaminofluorescein (DAF), to study both the physical and the chemical properties of stripped corn oil containing oleic acid and phosphatidylcholine. The fluorescence intensity of DAF increased with increasing water concentration in the edible oil. The addition of oleic acid decreased DAF fluorescence due to the ability of the free fatty acid to decrease the pH of the aqueous phase of the bulk oil. Phosphatidylcholine increased DAF fluorescence due to its ability to increase DAF exposure to the aqueous phase. Oleic acid had no impact on interactions between DAF and water-soluble peroxyl radicals, while phosphatidylcholine decreased peroxyl radical degradation of DAF. These results suggest that DAF could be a useful analytical tool to study the impact of the aqueous environment of bulk oil on lipid oxidation.