Porcine vasoactive intestinal polypeptide (VIP; 10(-9) to 10(-7) M) was a potent stimulator of prolactin (PRL) release by anterior pituitary cells from immature and laying turkey hens. Basal and VIP-induced PRL release of cells from laying hens were diminished (P less than 0.05) when the cells were cultured for 48 hr in the presence of charcoal-stripped laying hen serum, but not when the cells were cultured in the presence of whole laying hen serum. This change in VIP-induced PRL release was not evident when cells were derived from immature hens. Basal PRL release by cells from laying hens was not altered by the presence of estradiol (E2; 10(-12) to 10(-5) M), although such release was generally enhanced in cultures of cells from immature hens containing E2. The presence of E2 enhanced (P less than 0.05) the magnitude of the VIP-induced PRL release by cultures of cells from laying hens and diminished (P less than 0.05) the magnitude of this release in cultures of cells from immature hens. Cells from immature and laying hens exposed to progesterone (P4; 10(-5) M) for 96 hr exhibited enhanced basal PRL release, though lower P4 concentrations had no effect. Utilizing cells from laying hens, P4 exposure for 24 hr resulted in diminished (P less than 0.05) VIP-induced PRL release, while P4 exposure for 96 hr resulted in markedly enhanced (P less than 0.05) VIP-induced PRL release. Basal PRL release was generally not altered by the presence of testosterone (T). The VIP-induced PRL release by cells derived from immature and laying hens was diminished (P less than 0.05) by the presence of T. Prolactin release in the turkey is likely modulated by gonadal steroids acting directly on the cells of the anterior pituitary.