The effects of dopamine on PRL secretion and lysosomal enzyme activity in anterior pituitary tissue from rats selected during various stages of the estrous cycle were examined under in vitro conditions. During the estrous cycle, there was a marked variation in the capacity of dopamine to stimulate the activity of the lysosomal enzyme beta-glucuronidase in the anterior pituitary gland. Moreover, this variation in the responsiveness of pituitary tissue to the stimulatory action of dopamine on beta-glucuronidase activity was accompanied by a similar variation in the responsiveness of the tissue to the inhibitory action of dopamine on PRL release. Anterior pituitary glands from diestrous rats were the most sensitive to the actions of dopamine on beta-glucuronidase activity and PRL release, whereas glands from estrous animals were the least sensitive. Ovariectomy on the day of diestrus prevented the decline in the responsiveness of the anterior lobe to the actions of dopamine normally seen 2 days later (on the presumptive day of estrus). On the other hand, when animals were treated with estradiol benzoate during the 2 days after ovariectomy, the responsiveness of the pituitary tissue to dopamine was markedly suppressed and was similar to that of tissue from estrous rats. When rats were treated with progesterone during the 2 days after ovariectomy, the responsiveness of the anterior lobe to dopamine was similar to that in ovariectomized controls. It is suggested that the decrease in the responsiveness of the anterior pituitary gland to the actions of dopamine on lysosomal enzyme activity and PRL release that occurs between diestrus and estrus is estrogen mediated. It is also suggested that the ability of estrogen to antagonize the inhibitory effect of dopamine on PRL release may be mediated through an estrogen-induced reduction in the capacity of dopamine to stimulate lysosomal enzyme activity in the anterior pituitary gland.