Abstract The effect of nitrogen and carbon nutrition, growth and biomass storage on the effectiveness of microbial 9α-hydroxylation of 4-androstene-3,17-dione was studied. The transformation was carried out by resting Rhodococcus sp. cells in nutrient deficient transformation medium at different pHs as well as in the presence of several solvents of the steroid compounds. A conversion ratio of ca 0·75 was observed with stationary phase cells grown on medium containing glucose and non-dehydrated casein hydrolysate. A strong influence of the nitrogen source on the effectiveness of the process was noticed. The presence of different alcohols in the reaction medium was found to increase the substrate conversion ratio to ca 0·85. A decrease in the transformation ability of stored cells was a logarithmic function of the storage time.