Fundamental interfacial properties were determined at the hydrocarbon-water interface for 5 beta-cholanic acid, abietic acid, and three crude oil naphthenic acids separated from three naphthenic-acid-containing crude oils. Observed interfacial properties were correlated to the composition and chemical structure of crude oil naphthenic acids. Crude oil naphthenic acids are a mixture of primary, secondary, and tertiary, largely cycloparrafinic, organic acids in the molecular weight range of 250-750 amu. The percentage of primary naphthenic acids and proportion of low molecular weight naphthenic acids influence interfacial activity and emulsion stability of water-in-oil emulsions. Naphthenic acid composition and chemical structure determine the nature of molecular aggregation at the hydrocarbon-water interface and impact interfacial properties and emulsion stabilization performance. Crude oil naphthenic acids containing about 80% primary acids and a higher proportion of 250-425 molecular weight species were the most effective in reducing the interfacial tension and stabilizing water-in-oil emulsions. Medium effects on interfacial properties were investigated using a model hydrocarbon, 5:1: hexadecane:toluene, and naphthenic-acid-free crude oil as the hydrocarbon phase. Crude oil naphthenic acids reduce the interfacial tension of the naphthenic-acid-free crude oil to 18.5 dyn/cm, whereas they reduce the interfacial tension of the model oil to 28 dyn/cm. The enhanced activity of the naphthenic acids in the crude oil medium is attributed to the interaction between crude oil naphthenic acids and crude oil asphaltenes.