A dissociated preparation of normal adult rat pituitary cells has been used to study PRL autoregulation at the level of the mammotroph. Female rat pituitary cells previously cultured for 48 h on polylysine-coated petri dishes were washed to remove serum and accumulated PRL and then incubated in fresh medium in the absence or presence of increasing concentrations of rat PRL. Accurate balance sheets, allowing for degradation and nonspecific adsorption of PRL, showed exogenous PRL to regulate the amount of PRL released by the cells. That this regulation was partly produced by uptake of secreted PRL from the medium was demonstrated by supplementing the medium with [125I]iodo-rat PRL. Inhibition of secretion also played a role and was implied by experiments showing that ease of reversal of the inhibition was inversely proportional to the density of cell culture, which was itself proportional to the amount of PRL in the medium and the duration of autoregulation. These results indicate that normal adult rat pituitary cells in primary culture are capable of regulating the amount of PRL in their external milieu and that uptake of already secreted PRL is an important component of the regulatory mechanism.