Acid, basic and neutral fractions from Orinoco Belt Crude Oil (Cerro Negro) were isolated by ion-exchange chromatography and characterized by spectroscopic methods (FTIR, H- and 13C-NMR), titration with KOH and molecular weight measurements using vapor pressure osmometry (VPO). For the evaluation of the interfacial tension, a toluene/water model system was used. In general, it was determined that interfacially active compounds (natural surfactants) at high pH values are concentrated mainly in the acid fractions, strong acid fraction (SAF) and total acid fraction (TAF) of the crude oil. By interfacial tensions determinations, it was concluded that the hydrophobic portion of the natural surfactants has higher aromatic character and molecular weight whereas the hydrophilic region showed higher acidity and concentration of polar sustituents in comparison with the original Cerro Negro crude oil. Also it was found that SAF has lower interfacial activity per molecule than TAF. The results were explained in terms of a better hydrophilic-lipophilic balance of the TAF, (higher molecular weight, lower concentration of polar groups and higher total acid number) in comparison with the SAF. Finally, it was determined that Cerro Negro crude oil and its components suffer important changes (decarboxylation reactions, etc.) at temperatures normally reached during distillation ( > 250 degrees C), therefore, their interfacial activities are also substantially changed as well.