AbstractVaginae of newborn BALB/c mice were cultured in vitro with or without 1 μg/ml of estradiol for 1‐3 days, and then transplanted into 4‐day‐old syngeneic hosts. Intermittent or persistent estrus was indicated by smears from many of these transplanted vaginae which had previously been cultured with estradiol, even after ovariectomy of the hosts. Those transplants which had been cultured for 2‐3 days with estradiol had thickened epithelium (hyperplasia), and cornification was observed histologically in some. In contrast, where estradiol was not used in the cultures, transplanted vaginae (controls) showed no cornification after host ovariectomy. Histologically, the epithelium of these controls was thin and in most cases slightly mucified. Vaginae of 3‐week‐old BALB/c mice, cultured with or without estradiol, resembled the controls, derived from newborn donors.When vaginae from newborn donors were cultured with 0.01‐ 5 μg/ml of estradiol for three days, and then transplanted into 4‐day‐old syngeneic hosts, ovary‐independent hyperplasia and cornification were observed in a number of the transplants. When vaginae from newborn donors were cultured in 0.001 μg/ml of estradiol, or in 0.001‐5 μg/ml of testosterone, neither persistent cornification nor hyperplasia was observed subsequent to transplantation. These results suggest that the mechanism of induction of persistent vaginal estrus in vivo by estradiol and by testosterone may be different. Whereas estradiol may act directly on the vaginal epithelium of newborn mice, the effect of testosterone would appear to be indirectly mediated.