In this study, liquid culture systems containing rhamnolipid-solubilized, separate-phase, and multi-state hexadecane as the carbon source were employed for examining the effect of rhamnolipid solubilization on the bioavailability of hexadecane. Experimental results showed that the uptake of rhamnolipid-solubilized hexadecane by Pseudomonas aeruginosa ATCC 9027, a rhamnolipid producing strain, was enhanced compared to the uptake of mass hexadecane as a separate phase, indicating rhamnolipid solubilization increased the bioavailability of hexadecane for this bacterium. For Pseudomonas putida CICC 20575 which does not produce but degrade rhamnolipid, the uptake of either rhamnolipid-solubilized hexadecane or multi-state hexadecane was inhibited. The reduction of bioavailability was assumed to be the consequence of the blocking effect caused by the partition of rhamnolipid molecules at the hexadecane-water interface. The results show that how rhamnolipid solubilization changes the bioavailability of hexadecane depends on the bacterial compatibility to rhamnolipid. The study adds insight into the knowledge of biosurfactant-associated bioavailability of hydrophobic organic compounds (HOCs), and is of importance for application of biosurfactants in bioremediation of HOCs. (C) 2016 Elsevier B.V. All rights reserved.