Expression of relaxin is increased in human breast cancer, and relaxin was shown to increase in vitro invasiveness through increased production and secretion of matrix metalloproteinases in human breast cancer cells. The role of estrogen in the promotion of breast cancer is well-known. The environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a known carcinogen but has been shown to have antiestrogenic effects in human breast cancer cells. In this study, we have employed real-time PCR and chromatin immunoprecipitation (ChIP) assays to investigate the influence of estrogen and TCDD on relaxin-1 (RLN1) and relaxin-2 (RLN2) gene expression in MCF-7 and T47D human breast cancer cells. Estrogen increased RLN2 transcripts in T47D and MCF-7 cells after just 4 h of exposure, whereas TCDD did not. RLN1 transcripts were only induced after 24 h of estrogen exposure. TCDD did have antiestrogenic activity and reduced the estrogen-mediated increase in RLN2 and RLN1 mRNA. The estrogen-mediated increase in RLN2 mRNA levels was not caused by changes in the mRNA stability. ChIP analysis revealed binding of estrogen receptor-alpha (ERalpha) to promoter sequences of the RLN2 gene. Thus, we provide evidence that RLN2 gene activity is directly regulated by activated ERalpha in human breast cancer cells and we show that activation of the arylhydrocarbon receptor by TCDD inhibits this regulation by estrogen.