Although the importance of estradiol-17(E₂) in many physiological processes has been reported, no previous study has investigated the effects of E₂ on embryonic stem (ES) cell proliferation. In this study, therefore, we examined the effect of E₂ on the DNA synthesis of mouse ES cells and its related signaling pathways. The results of this study showed E₂ (10^-9 M) significantly increased [³H] thymidine incorporation at > 4 hr and E₂ (> 10^-12 M) induced an increase of [³H] thymidine incorporation following 8 hr incubation. Moreover, E₂ (> 10^-12 M) also increased BrdU incorporation and cell number. Indeed, E₂ stimulated estrogen receptors (ERand ER) protein levels and increased mRNA expression levels of protooncogenes (c-fos, c-jun, and c-myc). Tamoxifen (antiestrogen) completely inhibited E₂-induced increases of [³H] thymidine incorporation. In addition, E₂-BSA (10^-9 M) also increased [³H] thymidine incorporation at > 1 hr and E₂-BSA (> 10^-12 M) increased [³H] thymidine incorporation at 1 hr incubation. E₂-BSA-induced increase of BrdU incorporation also appeared in a dose-dependent manner. Tamoxifen had no effect on E₂-BSA-induced increase of [³H] thymidine incorporation. Also, E₂ and E₂-BSA displayed maximal phosphorylation of p44/42 MAPKs at 10 min or 5 min, respectively. E₂ increased cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2 and CDK 4. In contrast, E₂ decreased the levels of p21^cip1 and p27^kip1 (CDKs inhibitory proteins). Increases of these cell cycle regulators were blocked by PD 98059 (MEK inhibitor, 10^-5 M). Moreover, E₂-induced increase of [³H] thymidine incorporation was inhibited by PD 98059 or butyrolactone I (CDK 2 inhibitor). In conclusion, estradiol-17 stimulates DNA synthesis of mouse ES cells, and this action is mediated by MAPKs, CDKs, or protooncogenes.