AbstractChinese hamster V79 fibroblasts, frequently used as target cells in short‐term tests for mutagenicity, do not possess measurable monooxygenase activity; in particular, enzymatic oxidation of testosterone (T) cannot be demonstrated. If these V79 cells, however, had been transfected with the cDNA‐encoding rat liver cytochromeP‐450IIB1 under control of the SV40 early promoter, they stably expressed monooxygenase activity. These so‐called SD1 cells then oxidatively metabolized T at a rate of 27 pmol/mg protein/min, converting it to 16α‐ and 16β‐hydroxy‐T as well as 4‐androsten‐3,17‐dione as sole metabolites in a ratio of 1.1:1.0:1.6. The regio‐ and stereoselective conversion of T by SD1 cells, as well as the quantitative distribution of the metabolites, corresponds well with the results reported for pure cytochromeP‐450IIB1