Depending on the size and position of the substituent groups on the aromatic ring, the o-xylene dioxygenase from Rhodococcus sp. strain DK17 possesses the unique ability to perform distinct regioselective hydroxylations via differential positioning of substrates within the active site. The substrate-binding pocket of the DK17 o-xylene dioxygenase is large enough to accommodate bicyclics and can be divided into three regions (distal, central, and proximal), and hydrophobic interactions in the distal position are important for substrate binding. Current molecular and functional knowledge contribute insights into how to engineer this enzyme to create tailor-made properties for chemoenzymatic syntheses. (C) 2013 Elsevier Ltd. All rights reserved.