The estrogenic action of some persistent organochlorine pesticide residues may play a role in the progression of hormonally responsive tumors of the breast and uterus. The prototypical xenoestrogen o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT) acts by binding and activating the estrogen receptor (ER). The present study focuses attention on the mechanisms through which another organochlorine compound, beta-hexachlorocyclohexane (beta-HCH), exerts estrogen-like effects in human breast cancer cells. Both o,p'DDT and beta-HCH stimulated proliferation in a dose-dependent manner in the ER-positive cell lines MCF-7 and T47D but not in the ER-negative lines MDA-MB231, MDA-MB468, and HS578T. Both compounds produced an increase in the steady state level of pS2 mRNA in MCF-7 cells. These responses were equal in magnitude to the maximal effect of estradiol, and they were inhibited by inclusion of the antiestrogen ICI164384. On the other hand, when tested in a competitive binding assay, beta-HCH did not displace 17beta-[3H]estradiol from the ER even at a concentration that was 40,000-fold higher than the tracer steroid. Furthermore, nuclear retention of the ER during homogenization procedures was induced by a 2- or 24-h treatment of MCF-7 cells with o,p'-DDT and 17beta-estradiol but not by treatment with beta-HCH; this indicates that beta-HCH nether activates the ER, nor is it converted intracellularly to an ER ligand. Transcriptional activation by beta-HCH occurs in estrogen-responsive GH3 rat pituitary tumor cells transfected with a luciferase reporter construct driven by a complex 2500-bp portion of the PRL gene promoter; this trans-activation response is inhibited by inclusion of ICI164384. However, beta-HCH is ineffective in stimulating a reporter construct driven only by a consensus estrogen response element and a minimal promoter derived from the herpes simplex virus thymidine kinase gene. Thus, beta-HCH cannot act on a simple, single estrogen response element; rather, it requires the combinatorial regulation found in a complex promoter. These data are consistent with the notion that beta-HCH stimulation of cell proliferation and gene expression is ER dependent, but its action is not through the classic pathway of binding and activating the ER. beta-HCH may represent a new class of xenobiotic that produces estrogen-like effects through nonclassic mechanisms and, therefore, may be of concern with regard to breast and uterine cancer risk.