In order to test the in vivo effect of prolactin on estrogen receptor (ER) binding capacity in tumors induced by 7,12 dimethylbenz(a)anthrancene (DMBA-tumor), growth of the tumors from changes in prolactin and estrogen levels was compared retrospectively with cytoplasmic ER levels. It was demonstrated that some tumors required prolactin, some needed prolactin-estrogen during their growth period anda small number were not influenced by hormonal milieu. ER was present in hormonally dependent tumors but was low or absent in hormonaly-independent tumors. Deletion of hormones by endocrine ablation in the host rat resulted in tumor regression loss of ER. Replenishment of ER and subsequent tumor growth were accomplished by injection of prolactin or prolactin-estrogen in endocrine ablated rats but were not achieved in rats bearing tumors exposed to prolactin-nafoxidine. Our results demonstrate that both estrogen and prolactin were essential for growth of hormonally dependent DMBA-tumors. Tumor growth was also prevented when cytoplasmic ER was not replenished , indicating that ER may be an indispensable prerequisite for growth. Prolactin, independently of or cooperatively with estrogen, stimulated ER binding capacity. These results support the hypothesis that there may exist a prolactin regulatory mechanism of estrogen action at the tumor site. The interactions of estrogen and prolactin in situ in modulating hormonal receptor binding capacities may contribute to the overall stimulatory effect of these two hormones on DMBA-tumors.