17β-oestradiol (E2) may have a beneficial impact on the development of age-related diseases, in part through α and β oestrogen receptors (ER) in glia. Tibolone, a synthetic steroid, could influence glial-mediated neuroprotection if agonist oestrogenic activity is demonstrable. We used the N20.1 mouse oligodendrocyte cell line as a glial cell model to evaluate the response of ERα and ERβ through oestrogen-response element (ERE) and AP-1-driven reporters to E2, 4-hydroxytamoxifen (4OHT) and to two tibolone metabolites, 3α-hydroxytibolone (3α-OH-Tib) and 3β-hydroxytibolone (3β-OH-Tib). In addition, we tested the activity of these same ligands through the endogenous ERα in human normal astrocytes. Because endogenous ER was not detected in the N20.1 cells, we tested the ability of exogenous ER to activate transcription in response to ligands (100 nM) using a transient cotransfection assay with an ERα expression vector. To test the antagonist activity of 3α-OH-Tib and 3β-OH-Tib, we used them in combination with E2 (10−8 M), at concentrations of 10−7 M and 10−6 M. The human normal astrocytes were treated similarly, with the exception that no ER-encoding DNA was used. Specific ER ligand mediated activity was shown using the E2 antagonist ICI 182 780 and the pSG5 empty vector. E2, 3α-OH-Tib, and 3β-OH-Tib stimulated ERα on an ERE-promoter at each concentration (P < 0.001) but not at an AP-1-driven promoter. 4OHT was an effective antagonist, but did not exhibit agonist activity on the ERE-driven promoter. 4OHT was an effective agonist through ERα on an AP-1-driven promoter. 3α-OH-Tib and 3β-OH-Tib were not effective antagonists of E2. Both metabolites acted through the ER because the addition of an E2 antagonist blocked their activity. These results show that 3α-OH-Tib and 3β-OH-Tib exert agonist activity, yet lack antagonist or additive activity, through the ERα and ERβ on an ERE-driven but not on an AP-1-driven promoter in a glial cell model and in normal human astrocytes. This contrasts with the effects of 4OHT, which exerted little or no agonist activity, but reduced E2-stimulated activity through ERα on the ERE, in the same cells.