The actinomycete Rhodococcus jostii RHA1 contains a multitude of oxygenase enzymes, consonant with its remarkable activities in the catabolism of hydrophobic xenobiotic compds. In the interests of identifying activities for the transformation of drug mols., we have cloned genes encoding 23 cytochrome P 450 heme domains from R. jostii and expressed them as fusions with the P 450 reductase domain (RhfRED) of cytochrome P 450Rhf from Rhodococcus sp. NCIMB 9784. Fifteen of the fusions were expressed in the sol. fraction of Escherichia coli Rosetta (DE3) cells. Strains expressing the fusions of RhfRED with genes ro02604, ro04667, ro11069, ro11320, ro11277, ro08984 and ro04671 were challenged with 48 com. available drugs revealing many different activities commensurate with P 450-catalyzed hydroxylation and demethylation reactions. One recombinant strain, expressing the fusion of P 450 gene ro11069 (CYP257A1) with RhfRED, and named Ro07-RhfRED, catalyzed the N-demethylation of diltiazem and imipramine. This observation was in accord with previous reports of this enzyme’s activity as a demethylase of alkaloid substrates. Ro07-RhfRED was purified and characterized, and applied in cell-free biotransformations of imipramine (7 μM) giving a 63% conversion to the N-desmethyl product. [on SciFinder(R)]