In the present study, we used [3H]norethisterone to explore the bioconversion of this compound to A-ring reduced metabolites in African Green Monkey Kidney CV-1 cells and breast cancer T-47D cells. Additionally, we analyzed the capability of each norethisterone tetrahydro-reduced compound to bind the human oestrogen receptors alpha and beta and transactivate an oestrogen-sensitive reporter gene. The results showed that norethisterone is mainly metabolized to 3 alpha,5 alpha-norethisterone (>85% of total [3H]norethisterone added) by CV-1 and T-47D cells, and that both A-ring tetrahydro-reduced metabolites exhibit different capabilities to displace [3H]17beta-oestradiol from the oestrogen receptor alpha and beta, being 3 alpha,5 alpha-norethisterone the weakest competitor. We also found that 3 alpha,5 alpha-norethisterone and 3beta,5 alpha-norethisterone activate both oestrogen receptors at nanomolar concentrations and that the transactivation induced by the oestrogen receptor alpha was generally higher (1.7- to 4.0-fold) than that provoked by the beta receptor isoform. In oestrogen receptor alpha-transfected CV-1 and T-47 D cells, the oestrogenic-like potency of the 3beta,5 alpha-tetrahydro-reduced form was similar to that exhibited by 17beta-oestradiol and 2.5- to 4.0-fold higher than that shown by the 3 alpha,5 alpha-reduced compound; conversely, in the oestrogen receptor beta system the potency of the natural ligand was higher than that presented by the 3beta,5 alpha-tetrahydro-reduced metabolite. In CV-1 cells expressing the oestrogen receptor beta, the transactivation potency of 3beta,5 alpha-norethisterone was approximately 2-fold higher than that exhibited by its 3 alpha,5 alpha-tetrahydro-reduced isomer, whereas in T-47D cells the potency of the 3 alpha,5 alpha-tetrahydro-reduced compound was slightly higher than that shown by the 3beta,5 alpha A-ring reduced norethisterone metabolite. These results demonstrate that CV-1 and T-47D cells possess the enzymatic machinery to bioconvert norethisterone into the 5 alpha-reduced, 3 alpha-hydroxylated form and that neither 3 alpha,5 alpha- or 3beta,5 alpha-norethisterone exhibit preference or selectivity towards a particular oestrogen receptor isoform to induce a particular oestrogenic effect in these cell lines.