The purpose of this study was to decide the appropriate administration period in uterotrophic assay and to evaluate the usefulness of uterotrophic assay using two typical estrogen agonists and one antagonist, when chemicals were given orally. Diethylstilbestrol (DES), ethynyl estradiol (EE), or Atrazine (ATZ) was administered to immature Crj: CD (SD) IGS rats orally at doses of 0.01-1 μg/kg/day, 0.06-6 μg/kg/day, or 0.5-50 mg/kg/day from postnatal day (PND) 21 for 3 or 7 days, respectively. Additional groups of rats given ATZ orally at the above doses were also treated with 17β-estradiol (E₂) subcutaneously at a dose of 0.3 μg/rat/day. A vehicle control group given only olive oil was established in each study, and a group given only E₂ was included in the study of ATZ also. No abnormal clinical signs were detected in any of the groups. There were no significant differences in body weight between the vehicle control and each of the groups given DES, EE or ATZ, or the group given only E₂ and each of the groups given ATZ plus E₂. Premature vaginal opening was observed from 6 days in all rats given 6 μg/kg EE or ATZ plus E₂. Uterine fluid was also present in all rats given 6 μg/kg EE or ATZ plus E₂ for 3 or 7 days. Uterine weight was significantly higher in the rats given 1 μg/kg DES for 3 days and 6 μg/kg EE for 3 or 7 days, but no increases were detected in any of the rats given DES for 7 days. Although uterotrophic activity was not detected in any of the rats given only ATZ, co-treatment with 50 mg/kg ATZ and E2 for 3 days reduced the E₂-induced increases in uterine weight. The present results demonstrated that the appropriate period of oral administration for detecting the estrogenic properties of DES and EE or anti-estrogenic properties of ATZ using the immature rat uterotrophic assay was 3 days and ATZ exerted weak anti-estrogenic effects on the uterus.