Estrogen regulates various cytokines and growth factors in estrogen receptor (ER)-positive human breast cancer. Receptor activator of NF-kappaB ligand (RANKL) is an essential cytokine for osteoclasts, whereas osteoprotegerin (OPG) is a soluble inhibitor for RANKL. We analyzed the regulation of the RANKL/OPG system by estrogens and androgens in the ER-positive breast cancer cell line MCF-7 and the ER-negative breast cancer cell line MDA-MB-231. In MCF-7 cells, which predominantly express ER-alpha, 17beta-estradiol and testosterone dose-dependently decreased OPG mRNA levels and protein secretion by 70 and 65%, respectively (p<0.0001 by ANOVA). The inhibition of OPG production by 17beta-estradiol and testosterone was specifically prevented by the pure anti-estrogen ICI 182,780, and the testosterone effect was prevented by an aromatase inhibitor. In conclusion, 17beta-estradiol suppressed OPG production by human breast cancer cell lines in a dose-dependent and specific manner, indicating that the RANKL/OPG cytokine system is an estrogen-responsive target in breast cancer.