The growth of MTW9 mammary tumors exhibits different degrees of responsiveness to ovariectomy, ranging from sensitivity to resistance. This range of response is a function of time elapsing from tumor inoculation until performance of ovariectomy provided that prolactin (PRL) level is kept continuously high. In vivo studies showed that the MTW9 tumors developed by chronic administration of spiramide were sensitive to ovariectomy by 60 days, but they became resistant to ovariectomy by 100 days. However, when spiramide treatment was discontinued after tumor appearance, the tumors were still sensitive to ovariectomy by 100 days. Chromatofo‐cusing (CF) profile of cytosolic estrogen receptors (ER) correlated with the responsiveness to ovariectomy. A 2‐peak profile for tumors sensitive to ovariectomy, and only a one‐peak profile for tumors resistant to ovariectomy, were seen. Although the prolactin level in rats bearing the tumors was higher than in the normal rats, no correlation between the PRL level and the change in CF profile of ER over time was seen. Also, these changes could not be correlated with the tumor size. In vitro studies showed that incubation of cytosolic ER from a sensitive tumor (2 peaks) with PRL led to a CF profile with only one peak, characteristic of a resistant tumor. Leupeptin, molybdate and phenylmethylsulfonylfluoride (PMSF) could not prevent this transition. The effect was not reproduced by incubation with growth hormone or progesterone. Our data suggest that PRL, either directly or through intermediates, may play a role in changing the response to hormonal therapy of the mammary tumor MTW9.