Display omitted •CYP BM3 was used as a tool for whole-cell biotransformation of valuable drug metabolites.•CYP BM3 mutants regioselectively convert NET into 15β- or 16β-OH-NET.•E. coli is a better host organism than S. cerevisiae for in vivo NET bioconversion.•Resting cells produce higher amounts of NET metabolites than growing cells.•A large-scale fermentor setup for production of 15β- and 16β-OH-NET was developed. In the present study, the whole-cell biotransformation using strains expressing CYP BM3 mutants has been evaluated for the stereoselective hydroxylation of the steroid norethisterone (NET), a widely used contraceptive. First, an in vitro CYP BM3 mutant library screen was performed to identify mutants with high activity, as well asstereoselectivity. Subsequently, two different whole-cell setups (resting and growing cells) were tested in two different host organisms (Escherichia coli and Saccharomyces cerevisiae) expressing CYP BM3. It was found that resting E. coli whole cells produced the highest amounts of products and therefore this biocatalytic setup was further optimized for application in a laboratory-scale fermentor. In the optimized fermentor setup, high product yields (0.3g/L 15β-OH-NET and 0.16g/L 16β-OH-NET) were achieved while it was also shown that the regio- and stereoselectivities of the steroid hydroxylations, as determined during the in vitro library screen, were preserved in the whole-cell system. The combination of a mutant CYP BM3 library and the optimized whole-cell oxidation system represents a promising and cost-effective alternative to a wide range of in vitro biosynthetic routes.