Female BALB/c and B6C3F1mice were examined after a 3‐wk exposure to dietary estradiol (0, 400, 800, 1600, and 3200 ppb) in a purified (AIN‐76A) or a natural‐ingredient (NIH‐07) diet. The use of AIN‐76A was associated with a 9–13% greater (p < 0.001) body weight and a 36–43% higher (p < 0.001) serum cholesterol in both mouse genotypes when compared to mice fed NIH‐07. Conversely, when fed NIH‐07, both mouse genotypes had a 20–22% higher (p < 0.003) serum urea nitogren and 2–3.5% higher erythrocyte count (p < 0.001) and hemoglobin concentration (p < 0.04) than when fed AIN‐76A. Reduced erythrocyte parameters suggest that chronic feeding of the purified diet might result in anemia. No significant compound or diet‐related differences were noted for serum creatinine, alanine aminotransferase, aspartate aminotransferase, or gamma‐glutamyl transferase. Although there was no diet effect on absolute or differential white blood cell count, estradiol caused a decrease in the total white blood cell count (p < 0.014) and an increase in the percentage of poly‐morphonuclear leukocytes (p < 0.014) in BALB/c and decreased the percentage of lymphocytes (p < 0.005) in B6C3F1, females. In addition, estradiol increased uterine weight and inhibited thymic and splenic weights in one or both genotypes. Spleen and thymus weight responses to estradiol were not significantly influenced by diet. However, the uterine weight responses to estradiol were apparently influenced by diet in both genotypes. In B6C3F1mice, the uterus weighed more at each level of estradiol when mice were fed AIN‐76A compared to NIH‐07 diet. In BALB/c mice, this was true only at the two lower dietary concentrations of estradiol. In conclusion, mice fed the purified diet, AIN‐76A, differed from those fed the cereal‐based diet, NIH‐07, in hematology, clinical chemistry, and uterine weight response to estradiol.