Indigenous acidic crude oil compounds cause problems with regard to both the production and refining of crude oils. In this work, we have studied the molecular composition and interfacial properties of different acidic fractions. A North Sea acidic crude oil has been washed subsequently with pH 7, pH 10, and pH 14 aqueous solutions, resulting in three acidic fractions and three alkaline washed crude oils. The original crude oil, the acidic fractions, and the pH washed oils have been characterized by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FT- ICR MS) and by Fourier transform infrared spectroscopy. The emulsion stability of water- in- oil emulsions of the original crude oil and the alkaline washed crude oils has been determined by the critical electric field cell method. Further, the interfacial properties of the acidic fractions and the alkaline washed oils have been examined. FT- ICR MS shows that 90% of the acidic compounds from this crude oil consist of carboxylic acids, with molecular weights in the range 300800 Da. Removing the acidic compounds from the crude oils increases the interfacial tension and increases the water- in- oil emulsion stability, indicating that such indigenous acidic compounds destabilize water- in- oil emulsions.