The effects of dopamine (DA) on prolactin (PRL) secretion in anterior pituitary tissue from rats selected during various stages of the estrous cycle were analyzed under in vitro conditions. The results were examined in relation to plasma PRL, estradiol and progesterone levels. During the estrous cycle there was a marked variation in the responsiveness of the lactotrophs to the inhibitory action of DA. Rapid changes occurred during proestrus: pituitary lactotrophs were not sensitive to the inhibitory action of 10 n M DA at 15.00 and 17.00 h and were less sensitive to 1 µ M DA compared to 09.00 h (p < 0.01), 12.00 h (p < 0.05) and 19.00 h (p < 0.05). This lowest PRL response to DA was associated with the preovulatory PRL surge. The recovery of a higher PRL response at 19.00 h coincided with the decrease of plasma PRL levels. During the remainder of the cycle, plasma PRL levels remained low in estrus, diestrus 1 and diestrus 2. Lactotrophs were sensitive to 1 µ M and 10 n M DA during estrus and diestrus 2 but a significant lower PRL response to 1 µ M DA (p < 0.05) and no PRL response to 10 n M DA was observed in diestrus 1. These data show that alterations in the sensitivity of the lactotrophs’ responsiveness to DA occur in the anterior pituitary gland during the rat estrous cycle and can lead to the removal of DA inhibition in the presence of physiological DA concentrations. This phenomenon is consistent with the fact that DA could be involved in the preovulatory PRL surge during the estrous cycle.