Endothelin (ET), a recently discovered vasoconstrictor peptide, is widely distributed in different tissues including brain and pituitary. Although evidence regarding the role of ET in neuroendocrine processes is still fragmentary, it appears that the release of several anterior pituitary (AP) hormones can be modulated by peptides of the ET family. In the present study, we compared the effects of ET-1 and ET-3 on the release of PRL from AP cells cultured in serum-free (SFM) or serum-containing (SCM) medium. AP obtained from adult male rats were enzymatically dispersed, and the cells were plated in either SFM or SCM. After 4 days, cells were incubated with ET-1 or ET-3 for designated periods of time, and PRL levels in the incubation media were measured by RIA. When AP cells maintained in SCM were exposed to ET for 20 min, both peptides inhibited PRL release, with ET-1 being a more potent inhibitor than ET-3. In contrast, a biphasic response was observed in cultures grown in SFM: low concentrations of ETpeptides inhibited the secretion of PRL, while high concentrations caused a significant stimulation. Further characterization of the effects of ET-1 revealed that the stimulatory phase was relatively short (15–30 min) and was followed by inhibition of PRL release. The addition of either horse serum or fetal bovine serum to SFM on the day of plating resulted in a dose-dependent reduction in the ET-induced stimulation of lactotrophs. These findings indicate that the presence of serum in culture medium alters the expression of cell properties underlying inhibitory and stimulatory responses to ET in terms of PRL secretion. The biphasic response of lactotrophs to ET under serum-free conditions may be a result of paracrine interactions between different cell populations within the AP. Furthermore, regardless of the presence or absence of serum in culture medium, ET-1 is more effective than ET-3 in modulating PRL release.