Background: Combined hormone treatments in post-menopausal women have different clinical responses on uterus and vagina; therefore, we investigated differences in steroid signalling between various hormone therapies in these tissues. Methods: A total of 30 post-menopausal women scheduled for hysterectomy were distributed into four subgroups: control-group (n = 9), Tibolone-group (n= 8); estradiol (E-2)-group (n = 7); E-2 + medroxyprogesterone acetate (MPA)-group (n = 6). Medication was administered orally every day for 21 days prior to removal of uterus and upper part of the vagina. Tissue RNA was isolated, and gene expression profiles were generated using GeneChip technology and analysed by cluster analysis and significance analysis of microarrays. Apoptosis and cell proliferation assays, as well as immunohistochemistry for hormone receptors were performed. Results: 21-days of treatment with E-2, E-2 + MPA or tibolone imposes clear differential gene expression profiles on endometrium and myometrium. Treatment with E-2 only results in the most pronounced effect on gene expression (up to 1493 genes differentially expressed), proliferation and apoptosis. Tibolone, potentially metabolized to both estrogenic and progestagenic metabolites, shows some resemblance to E-2 signalling in the endometrium and, in contrast, shows significant resemblance to E-2 + MPA signalling in the myometrium. In the vagina the situation is entirely different; all three hormonal treatments result in regulation of a small number (4-73) of genes, in comparison to signalling in endometrium and myometrium. Conclusions: Endometrium and myometrium differentially respond to the hormone therapies and use completely different sets of genes to regulate similar biological processes, while in this experiment the upper part of the vagina is hardly hormone responsive.