The aim of this study was to test the hypothesis that some of the proliferative effects of steroid hormones on cancer cells are mediated by the Raf proto-oncogenes. The human breast cancer cell line MCF-7 is estrogen-receptor (ER) positive (+). NCI/ADR-RES is a human cell line lacking the estrogen receptor (ER-) that was initially named MCF-ADR. Raf-1, A-Raf and B-Raf kinase activities were examined in cell lines treated with beta-estradiol for 24 hours. Increases in Raf-1 and A-Raf activities were observed after treatment with beta-estradiol in the ER (+) MCF-7 cells but not in the ER (-) NCI/ADR-RES cells. In contrast, no significant changes in B-Raf activity were observed. Thus beta-estradiol can induce Raf-1 and A-Raf activities in ER (+) cells. In addition, beta-estradiol caused cell cycle progression in MCF-7 cells and an increased proliferative response to beta-estradiol was observed in MCF-7, which overexpressed constitutively-active Raf-1 (MCF/DeltaRaf-1). Increased mRNA levels of the ligand for the c-erb-B2 receptor, amphiregulin (ARG) were observed after beta-estradiol treatment of MCF-7 cells whereas constitutively higher levels of ARG and its receptor, c-erb-B2 mRNAs were detected in MCF/DeltaRaf-1 cells. These findings suggest that targeting Raf may prove efficacious in breast cancer therapies.