The hypothalamic peptides pituitary adenylate cyclase-activating polypeptides (PACAPs) may play central roles in the regulation of anterior pituitary gland function. The two pro-PACAP-derived peptides, PACAP38 and PACAP27, are posttranslationally processed from a common precursor molecule and share amino acid sequence homology with vasoactive intestinal peptide (VIP). To further evaluate and compare the physiological roles of these peptides, we have examined the short and long term effects of PACAP38, PACAP27, and VIP on POMC-related peptide production in the mouse AtT-20/D16v corticotrope cell line. Short term (0.5- to 5-h) treatment of AtT-20/D16v cells with PACAP38, PACAP27, or VIP elicited similar concentration-dependent and biphasic stimulation of ACTH release. Half-maximal stimulation of the higher potency phase was attained with 1-2 nM peptide; maximal secretion was observed at peptide concentrations greater than 100 nM. Similar 2- to 3-fold maximal stimulation of ACTH secretion was elicited by all three bioactive peptides; linear sustained ACTH secretion was observed. Long term (12- to 72-h) treatment of AtT-20/D16v cultures with these peptides resulted in substantially decreased rates of cellular division, with a concomitant increase in cell size and formation of cell processes characteristic of cellular differentiation. These morphological changes coincided with a sustained 2-fold increase in AtT-20 corticotrope intracellular hormone content and secretion. Northern blot analysis demonstrated a parallel induction of POMC mRNA expression by the PACAP38, PACAP27, and VIP peptides. These results suggest that AtT-20/D16v cells possess primarily the type II PACAP receptor subtype, which binds PACAP38, PACAP27, and VIP with apparent equal affinity. Furthermore, the long term effects of these peptides implicate a potentially significant role for PACAP and VIP peptides in the mediation of altered pituitary gland functions. In this vein, the PACAPs may prove to be unique regulators of neuroendocrine function and development.