Estrogen-binding proteins have been characterized in variants of the MTW-9B rat mammary tumor in an attempt to determine the functional significance of the low affinity cytosolic estrogen binder. In vivo selection of tumor variants was carried out by transplant of the tumor into intact and castrated male and female Wistar-Furth rats for four or five successive transplant generations. Tumors that developed in each of the four lines were taken for retransplant into intact and castrated male and female rats and all recipient tumor groups were compared for high and low affinity estrogen-binding proteins using isoelectric focusing analysis. No alterations in the isoelectric focusing profiles were observed in tumors that developed after repeated passage in ovariectomized female or castrated male rats when compared to the profile of the estrogen-binding proteins of the parent tumor carried routinely in intact female rats. However, a tumor variant containing only the low affinity, more basic estrogen-binding protein resulted from repeated passage of the MTW-9B mammary tumor in male rats. The high affinity estrogen receptor was absent in this variant and could not be induced by retransplant of the tumor into intact female hosts. Growth of the parent tumor and each of the variants was shown to be ovarian independent, suggesting that the presence of the low affinity estrogen-binding protein is not predictive of estrogen responsiveness. This suggestion is further supported by the observation that estrogen-stimulated progesterone receptor synthesis could be demonstrated in the parent tumor which demonstrated both binders, but not in the tumor variant developed in male rats which contained only the low affinity estrogen-binding protein. Progesterone receptor synthesis in this latter tumor appeared to be constitutive. Studies are continuing in an attempt to identify a role for the cytosolic low affinity estrogen-binding protein.