Estrogens are thought to regulate female reproductive functions by altering gene transcription in target organs primarily via the nuclear estrogen receptor-α (ER-α). By using ER-α “knock-out” (ERKO) mice, we demonstrate herein that a catecholestrogen, 4-hydroxyestradiol-17β (4-OH-E₂), and an environmental estrogen, chlordecone (kepone), up-regulate the uterine expression of an estrogen-responsive gene, lactoferrin (LF), independent of ER-α. A primary estrogen, estradiol-17β (E₂), did not induce this LF response. An estrogen receptor antagonist, ICI-182,780, or E₂ failed to inhibit uterine LF gene expression induced by 4-OH-E₂ or kepone in ERKO mice, which suggests that this estrogen signaling pathway is independent of both ER-α and the recently cloned ER-β. 4-OH-E₂, but not E₂, also stimulated increases in uterine water imbibition and macromolecule uptake in ovariectomized ERKO mice. The results strongly imply the presence of a distinct estrogen-signaling pathway in the mouse uterus that mediates the effects of both physiological and environmental estrogens. This estrogen response pathway will have profound implications for our understanding of the physiology and pathophysiology of female sex steroid hormone actions in target organs.