Evidence exists for expression of estrogen receptor β (ERβ) in human colonic mucosa. Here we investigated the expression of the classical ER (ERα) and of four isoforms of the human ERβ in HCT116, HCT8, DLD-1, and LoVo colon adenocarcinoma cell lines. In addition, [³H]17β-estradiol (17βE₂) binding to intact colon cancer cells was evaluated. RT-PCR and Western blot analyses showed lack of expression of the classical ERα in the four colon cancer cell lines. Conversely, wild-type ERβ isoform 1 was highly expressed in HCT8, HCT116, DLD-1, and LoVo cells and isoforms ERβ2–5 were present in HCT8 and HCT116 cells. Scatchard and Hill analysis of [³H]17βE₂ binding to the four different colon cancer cells revealed the presence of two classes of binding sites, one with high affinity (K_d values of 1–2 nM) and the other with lower affinity (K_d values of 10–20 nM). Forty-eight hour-pretreatment of cells with 1 and 10 nM 17βE₂ did not induce an increase of progesterone-specific binding to HCT8 cells, while a significant induction was observed after treatment with 10 nM 17βE₂ in HCT116 and DLD-1 cells and with both concentrations in LoVo cells. In addition, 1 pM–0.1 nM 17βE₂ significantly induced cell proliferation of HCT8 cells, while reducing growth of HCT116 and DLD1 cells at 10 nM–1 μM concentrations and of LoVo cells at all tested concentrations (1 pM–1 μM). These in vitro findings pose the basis for in vivo functions of ERβ and ERβ-interacting molecules in human colon cancer tissue.