Toluene 4-monooxygenase is a four-protein component diiron enzyme complex. The enzyme catalyzes the hydroxylation of toluene to give p-cresol with similar to 96% regioselectivity. The performance of the enzyme in two-phase reaction systems consisting of toluene, hexane, or perfluorohexane and an aqueous buffer was rested. In each of the cosolvent systems, containing up to 93% (v/v) of solvent, the enzyme was active and exhibited regioselectivity indistinguishable from the aqueous reaction. Using the perfluorohexane/buffer system, a number of polycyclic aromatic hydrocarbons were oxidized that were not readily oxidized in aqueous buffer. An instability of the hydroxylase component and a substantial uncoupling of NADH utilization and product formation were observed in reactions that were continued for longer than similar to3 min. More stable enzyme complexes will be needed for broad applicability of this hydroxylating system in nonaqueous media.