The use of organic solvent-based media for microbial 9 alpha-hydroxylation of androstenedione (AD) by resting Rhodococcus sp. cells was evaluated. Product yields higher than 90% were observed in a two-phase phthalate-buffer transformation medium (3: 1, v/v) containing 1.0 g L-1 AD. The results suggest that in these conditions the reaction of Delta(1)-dehydrogenation is considerably inhibited. The successful hydroxylation reaction with a product yield exceeding 60% was found to take place also in a phthalate medium under simultaneous forced aeration and maintenance of a minimum water content of about 3% (v/v). (c) 2005 Elsevier Inc. All rights reserved.