Several strands of evidence indicate that oestrogens exert a protective role against the development of colon cancer through indirect and direct effects on colonic epithelium. Oestrogen receptor beta (ERbeta), the predominant ER subtype in human colon, is significantly decreased in colonic tumours compared with normal mucosa suggesting a potential role in the regulation of colon tumour growth. To investigate this hypothesis we engineered human colon cancer ERalpha-negative HCT8 cells in order to obtain ERbeta protein over-expression. Stably transfected cells were cloned and ERbeta expression and functionality were monitored by RT-PCR, Western blotting and transactivation in an assay using oestrogen-responsive reporter constructs. Over-expression of ERbeta inhibited cell proliferation and increased cell adhesion in a ligand-independent manner. Its constitutive activation is possibly due to cross-talk with intracellular signalling pathways, as epidermal growth factor and IGF-I were able to induce ERbeta transactivation. A possible mechanism by which ERbeta over-expression inhibits proliferation in HCT8 cells is by modulation of some key regulators of the cell cycle; there is a decrease in cyclin E and an increase in the cdk inhibitor p21CIP1. In fact, flow cytometry analysis provided evidence for blocking of the G1-S phase progression induced by ERbeta over-expression. The magnitude of this effect was affected by the level of ERbeta expression. These results provide the first direct evidence that ERbeta plays an important role in colon cancer as a regulator of cell proliferation through the control of key cell cycle modulators and arrest in G1-S phase transition. These findings are compatible with the hypothesis that the loss of ERbeta expression could be one of the events involved in the development or progression of colon cancer.