A eucaryotic cell assay was established to identify novel, dual and selective inhibitors of human 5alpha-reductase. For this purpose the cDNAs encoding 5alpha-reductase type I and type II were inserted into a pRcCMV vector and expressed in human embryonic kidney (HEK293) cells. Single cell clones with substantially high enzymatic activity were selected and established as permanent cell lines. KM values were determined for both isozymes. The inhibitory potency of several steroidal and non-steroidal compounds synthesized in our group, as well as finasteride and 4MA as controls, were tested by measuring the conversion of [3H]androstenedione. Reaction products were quantified by a HPLC reversed phase technique. Using the new cell assays, selective as well as novel dual 5alpha-reductase inhibitors with IC50 values between 1.0 and 2.5 microM were identified.