The in vivo and in vitro studies conducted to examine whether E2 functions as an initiator or a promoter in mammary carcinogenesis can be summarized as follows: (1) Clinical and animal studies in vivo have shown a positive correlation of up-regulation of E2 C16 alpha-hydroxylation with either the presence of or the risk for breast cancer, suggesting that this metabolic alteration may represent an early-occurring event in the multistep process of tumorigenesis. (2) The mammary tissue, target for carcinogenesis, exhibits cancer risk-dependent alteration in E2 metabolism in the rodent and human mammary explant culture model, indicating that E2 metabolites may directly influence the mammary epithelium. (3) The 16 alpha-hydroxylated metabolite of E2, 16 alpha-OHE1, induces genotoxic DNA damage and aberrant hyperproliferation similar to that induced by chemical carcinogens in the rodent cell culture model. In preinitiated or fully transformed rodent or human cells, 16 alpha-OHE1 promotes the expression of transformed phenotype. (4) The initiator-mediated perturbation of E2 C16 alpha-hydroxylation in rodent and human mammary explant cultures is modulated by naturally occurring dietary constituents that are known to modulate rodent mammary tumorigenesis. (5) The observed effect of E2 on mammary tumorigenesis may be due in part to the generation of 16 alpha-OHE1, which functions as a weak initiator or a potent promoter of tumorigenic transformation in mammary epithelial cells. (6) The reaction of 16 alpha-OHE1 with the transcription factor ER is unique in that it can be irreversible and leads to aberrant gene expression.