A two-phase partitioning bioreactor was considered to remove toluene contained in a biodegradable organic phase by activated sludge (AS). The selected solvent was hexadecane. In a first step, the biodegradation of toluene dissolved in hexadecane by AS was examined. In a second step, acclimation of the AS was carried out in order to improve the biodegradation rate. Acclimation improved toluene removal, since biodegradation yield increased from 72% to more than 91%. A total consumption was observed after only 4 days culture with acclimated AS, since the rest of the toluene corresponded to gas leak; while in the case of non-acclimated sludge, losses cannot account for all non-degraded toluene. Regarding hexadecane, acclimation also improved its degradation, from 43% to 79% after 6 days culture for non-acclimated and acclimated AS, respectively.