Epidemiological and animal studies have suggested that chronic alcohol consumption is a major risk factor for osteoporosis. Using bone from cycling female rats infused chronically with ethanol (EtOH) in vivo and osteoblastic cells in vitro, we found that EtOH significantly increased estrogen receptor alpha (ERalpha) and beta (ERbeta) mRNA and ERalpha protein levels. Treatment with 17beta-estradiol (E2) in vivo and in vitro interfered with these effects of EtOH on bone and osteoblastic cells. ERalpha agonist propylpyrazoletriol (PPT) and ERbeta agonist diarylpropionitrile (DPN) attenuated EtOH-induced ERalpha and ERbeta gene overexpression, respectively. Similar to the ER antagonist ICI 182780, EtOH blocked nuclear translocation of ERalpha-ECFP in the presence of E2 in UMR-106 osteoblastic cells. EtOH also downregulated ERE-luc reporter activity. On the other hand, EtOH by itself upregulated some common ERalpha- and ERbeta-mediated genes apparently by an ER-independent pathway. EtOH also transactivated the luciferase activity of the p21 promoter region independent of additional exogenous ERalpha, activated p21 and p53, and stimulated senescence-associated beta-galactosidase activity in rat stromal osteoblasts. E2 treatment attenuated these EtOH actions. We conclude that inhibitory cross-talk between EtOH and E2 in osteoblasts on ERs, p53/p21, and cell senescence provides a pathophysiologic mechanism underlying bone loss and the protective effects of estrogens in alcohol-exposed females.