The surface active properties of highly purified glycolipid (rhamnolipid biosurfactants) of known structure have been studied in detail and their properties are reported. Interfacial tensions (TFT) against different classes of hydrocarbons have been measured experimentally by interfacial tensiometer measurement. The critical micelle concentration (cmc), maximum interfacial excess concentration (Gamma (max)), and the minimum area per headgroup (Amin) can be calculated from the IFT data. Furthermore, the free energy upon adsorption on the interface (DeltaG(ad)) and the free energy of surfactant to form micelle (DeltaG(mic)) can be derived from Gamma (max),A(min) and the cmc. The effectiveness and the distinct properties of micelles formed in the presence of linear, branched, and nomoaromatic solutes was evidenced by lower interfacial tensions against the linear alkanes (Gamma (min)) and a smaller area per headgroup (A(min)). Traditional explanations for this micellization behavior focus on more effective intercalation oflinear alkanes into the micelle core as opposed to localization of the more hydrophilic aromatic solutes in the palasades region of the micelle. Both rhamnolipid biosurfactants exhibited the smallest Amin and lowest interfacial tension and the lowest DeltaG(mic) in the presence of linear alkanes. The branched alkane hydrocarbons exhibited similarly small A(min) and IFTmin. The observed differences between the PG201 and Dyna270 rhamnolipid may be accounted for similarly to those observed in ionic systems, where substantially different cmc values are observed for different surfactants containing the same alkyl chain length but different headgroups. PG201 produced rhamnolipid contains a higher fraction of rhamnolipid RI which possess a much larger dirhamnosyl headgroup. Differences in the area per headgroup and other physical parameters determined are attributed to these differences in biosurfactant headgroup structure.