We reported previously that both subtypes of estrogen receptors, ERalpha and ERbeta, are expressed by human urothelial cells and mediate estrogen-induced cell proliferation in these cells. The aim of this study was to determine the extent to which each ER subtype contributes to urothelial cell proliferation and their possible involvement in the regulation of the cell cycle. We compared the expression of ERalpha and ERbeta mRNAs and protein quantitatively in primarily cultured human bladder urothelial cells obtained from six individuals with three immortalized urothelial (E6, E7, and UROtsa) and two bladder cancer cell lines (HTB-9 and T24). We found that all these cells express similar levels of ERbeta, but immortalized and cancer cells express much higher amounts of ERalpha than primary cells. Higher levels of ERalpha mRNA were also observed in the biopsies of bladder transitional cell carcinoma compared with sample from the same bladder unaffected by tumor. Using the ERalpha-selective agonist PPT, the ERbeta-selective agonist DPN, and specific small interfering RNA against ERalpha or ERbeta, we found that ERbeta predominantly mediates estrogen-induced G1/S transition and cell proliferation in the primary urothelial cells. By contrast, ERalpha predominantly mediates estrogen-induced G1/S transition and cell proliferation in bladder cancer cell lines. Furthermore, we found that 17beta-estradiol (E(2)) rapidly induces phosphorylation of extracellular signal-regulated kinases, but U0126, a mitogen-activated protein kinase kinase (MEK) inhibitor, does not affect E(2)-induced urothelial cell proliferation. E(2) up-regulated cyclin D1 and cyclin E expression in both the primary and bladder cancer cells, and the cancer cells have higher cyclin D1 and cyclin E expression during G0/G1 phases. Our data suggest that estrogen exerts its effects through different ER subtypes in urothelial cells. Increased expression of ERalpha may contribute to early induction of cyclin D1 and cyclin E during the cell cycle in bladder cancer cells.