The effect of pertussis toxin (PTX) pretreatment on endothelin-3 (ET-3)-mediated inhibition of prolactin secretion from primary cultures of rat anterior pituitary cells was examined. Monolayer cultures of anterior pituitary cells were treated with either 20 ng/ml PTX dissolved in media or with media alone (control) on the third day of culture. Exactly 24 h after PTX pretreatment, cells were challenged with either 100 nM ET-3 dissolved in media or media alone (control) for 4 or 48 h. ET-3 significantly (P less than 0.01) inhibited prolactin secretion in both the 4 and 48 h incubations. However, if the cells had been previously treated with PTX, ET-3 did not significantly affect prolactin secretion. These data suggest that a PTX-sensitive G protein mediates ET-3-induced inhibition of prolactin secretion and that ET-3 may invoke a signal transduction mechanism in the lactotroph which is distinct from those described in other cell types.