The impact of 17beta-estradiol and antiestrogens on uterine cancer cells is poorly understood. The aim of this study was to determine the impact of 17beta-estradiol, 4-hydroxytamoxifen, raloxifene and ICI 182 780 on the cell proliferation of six uterine cancer cell lines: HeLa, HEC-1-A, KLE, RL-95-2, Ishikawa and EN-1078D. The effects of these compounds on the cell proliferation of the six uterine cancer cell lines were studied in the presence and absence of estrogens (phenol red and serum deprivation of sex steroids). In a general manner, 17beta-estradiol and 4-hydroxytamoxifen showed similarities in their effects whereas raloxifene showed a different pattern of cell proliferation (agonistic and antagonistic) and ICI 182 780 had antagonistic activity. In the presence and absence of estrogens, we observed that each cell line had diverse expression of ERalpha, ERbeta, GPR30 and REA. GPR30 mRNA expression was significantly reduced in a serum/phenol-free medium. REA mRNA expression was not influenced by the media. Results demonstrated the importance of removing phenol red and the use of deprived serum when studying uterine cancer cells in relationship with 17beta-estradiol and antiestrogens. The affinity of each compound to the binding of ERalpha and ERbeta was very similar with the exception of raloxifene that had a preference for ERalpha binding. Akt phosphorylation/activity was reduced in cells cultured in a phenol red- and steroid-free culture medium indicating that the presence of steroids in the culture media can influence the activity of this survival pathway. Our results suggest that the expression of ERalpha, ERbeta and GPR30 are influenced by sex steroids and might play a role in the response of cells to 17beta-estradiol and antiestrogens but are not the only factors involved in this process.