This study describes an efficient, large scale fermentation of a recombinant alpha-L-rhamnosidase originating from Aspergillus terreus. High-cell-density Pichia pastoris fermentation resulted in yields up to 627 U/L/h. The recombinant enzyme was used for the reverse rhamnosylation of various small organic compounds. A full factorial experimental design setup was applied to identify the importance of temperature, substrate concentrations, solvent type and concentration as well as the acidity of the reaction mixture. Careful optimization of these parameters allowed the synthesis of a range of alpha-L-rhamnosides among which cyclohexyl alpha-L-rhamnopyranoside, anisyl alpha-L-rhamnopyranoside and 2-phenylethyl alpha-L-rhamnopyranoside. In addition, alpha-L-rhamnosylation of phenolic hydroxyls in phenols such as hydroquinone, resorcinol, catechol and phenol was observed, which is a rather unique reaction catalyzed by glycosidases. (C) 2013 Elsevier Ltd. All rights reserved.