The association between treatment of pregnant women with diethylstilbestrol (DES) and reproductive tract abnormalities in their female offspring is well known. Reports of comparable in utero exposure in animals are few. In this paper, pregnant outbred mice were treated s.c. with daily doses of DES ranging from 0.01 to 100 µg/kg on Days 9 to 16 of gestation. This period corresponds with major organogenesis of the reproductive tract in the mouse. Additional groups of pregnant mice were treated during the same time interval with 100-µg/kg doses of either dimethylstilbestrol (DMS), a weakly estrogenic estructural analog of DES, or 17β-estradiol, a potent steroidal estrogen. When female offspring of mice gestationally exposed to DES were sacrificed at 12 to 18 months of age, lesions were found throughout their reproductive tracts. The vagina was characterized by excess keratinization and female hypospadias, and, at the dose of 100 µg/kg, 5 of 20 mice had epidermoid tumors of the vagina; in one of the 35 mice derived from mothers treated with DES (10 µg/kg), vaginal adenocarcinoma was observed. The cervix in the DES-exposed offspring was enlarged, even though the size of its lumen was not different from that in the controls. Stromal stimulation of the cervix was apparent, and a low incidence of benign (leiomyoma, papilloma) as well as malignant (stromal cell sarcoma, leiomyosarcoma) tumors were seen. There was evidence of stimulation in the epithelium, and stroma of the uterus and cystic endometrial hyperplasia was common; a low incidence of benign (leiomyomas) and malignant (adenocarcinoma, stromal cell sarcoma) tumors was observed. The ovaries of prenatally DES-treated females were cystic more often than were those of the corresponding controls; at the highest dose, three ovarian tumors were noted, and the fallopian tubes were inflamed and congenitally malformed. Genital tract tumors were not seen in 85 control females or in the 12 or 10 females exposed prenatally to estradiol or DMS, respectively. In fact, in 12- to 13-month-old females derived from mice exposed during pregnancy to DES (100 µg/kg), common findings, which were absent or in very low incidence in control, estradiol-treated, or DMS-treated mice, included: vaginal concretions and excess keratinization; cervical enlargement; uterine squamous metaplasia and cystic endometrial hyperplasia; and oviductal malformations. The differences in effects among DES, estradiol, and DMS may be linked to differences in relative bioavailability of the compounds to the fetal target tissue. The results presented suggest a role for different embryonic rudiments as well as for tissue components in the observed long-term effects of gestational exposure to DES on the female reproductive tract. Moreover, such prenatal studies with DES in mice may be helpful in understanding the role of estrogens in the functional development of the genital tract and may ultimately provide a useful experimental model.