This study evaluates the transcriptional and post-transcriptional regulation of prolactin (PRL) by vasoactive intestinal peptide (VIP). Pituitary nuclei from laying (control), incubating (with enhanced VIP secretion), and VIP-immunized laying turkey hens, and from pituitary cells cultured with or without VIP were used in nuclear run-on transcription assays. Cytoplasmic PRL mRNA was analyzed by slot blot hybridization. PRL transcription was greater in hyperprolactinemic incubating birds (PRL/β- actin=3.33) than in laying birds (PRL/β-actin=1.83). VIP-immunoneutralized birds had 47% and 51% decreases in PRL transcription and cytoplasmic PRL mRNA, respectively when compared with laying birds. In primary pituitary cell cultures, VIP significantly increased the transcription rate of PRL (3.8- fold) and cytoplasmic PRL mRNA (3.2-fold) compared with that of non-VIP- treated pituitary cells. The stability of pre-existing PRL mRNA was measured by Northern blot analysis after addition of actinomycin D. PRL mRNA half, lives were calculated using a two-component model, with a first-long component of 18.0 ± 1.0 h and a second-short component of 3.7 ± 0.7 h in non-VIP-treated pituitary cells. Both half-lives were significantly increased (53.2 ± 6.9 and 26.3 ± 4.3 h) in VIP-treated cells. The present data show that VIP acts to stimulate PRL expression by up-regulating the transcription rate of PRL and by enhancing PRL mRNA stability.