Female ACI rats were exposed to diethylstilbestrol (DES) in utero to evaluate the effects on the peri-pubertal mammary gland with respect to 1) mammary gland morphology, 2) sensitivity to natural and synthetic estrogens, and 3) sensitivity to endogenous epidermal growth factor (EGF). Pregnant rats were injected with vehicle (sesame oil) or DES (total dose, 8.0 micrograms) on days 15 and 18 of gestation. DES-exposed and control offspring were ovariectomized at 34 days of age and sacrificed at day 53 to ascertain the morphology of the mammary glands in peri-pubertal rats. Elvax pellets containing 5 or 11 ng 17 beta-estradiol (E2) or DES were implanted subcutaneously adjacent to the third mammary gland pair. Furthermore, additional groups of rats were subjected to bilateral sialoadenectomy at the day of ovariectomy to remove the major source of endogenous EGF. A significant proportion of mammary glands of DES-exposed animals exhibited atypical mammary gland morphology, with approximately 25% displaying hypo-differentiation, and about 5% with aberrant hyper-proliferation. From the pellet implantation experiments, the DES-exposed glands were found to be refractory to stimulation by 5 and 11 ng DES; however, there was no significant difference in the degree of local stimulation elicited by either dose of E2. Sialoadenectomy at d34 had no apparent effect on mammary gland morphology in either the DES-exposed or vehicle-exposed groups. These data support the premise that the mammary gland of the peri-pubertal ACI rat is morphologically and physiologically aberrant as a function of transplacental exposure to DES, with a significant percentage hypo-differentiated and refractory to subsequent hormonal stimulation.