Various environmental chemicals, both natural and synthetic, are believed to act as endocrine disruptors (EDs) in mammals. In this study, a new in vivo model of immature rats was used to explore the induction of calbindin-D9k (CaBP-9k) following a single injection of EDs. In a time-dependent experiment, immature rats at postnatal day 16 were treated with high doses (600 mg/kg body weight [BW]) of 4-tert-octyphenol (OP), p-nonylphenol (NP), or bisphenol A (BPA), and euthanized at different time points (3, 6, 12, 24, or 48 h). For a dose-dependent study, immature rats were given different doses (200, 400, or 600 mg/kg BW) and euthanized at 24 h after injection. After treatment with these EDs, the effects on CaBP-9k mRNA and protein were examined by Northern and Western blot analyses, respectively. An anti-estrogen, ICI 182,780, was employed to examine the potential involvement of estrogen receptor (ER) in the induction of estrogen receptor-mediated physiologic responses in vivo. A single treatment with each of the chemicals, at 600 mg/kg BW, resulted in a significant increase in the expression of CaBP-9k mRNA and protein 24 h after injection. In addition, treatment with OP, NP, or BPA resulted in a positive uterotrophic response. Cotreatment with the ER antagonist ICI 182,780 completely prevented the ED-induced uterine weight gain. Taken together, these results demonstrate that a single injection of OP, NP, or BPA results in an increase of CaBP-9k mRNA and protein via an ER-dependent pathway in the uterus of immature rats. This new model may be important to elucidate the mechanism of action of xenoestrogens on estrogen-sensitive tissue.