Pituitary adenylate cyclase-activating polypeptide (PACAP) has been proposed to be the ancestral growth hormone (GH)-releasing hormone. Recently, using grass carp as a model for modern-day bony fish, we have demonstrated that PACAP nerve fibers are present in close proximity to carp somatotrophs and mammalian PACAPs can induce GH secretion in carp pituitary cells. To further examine the role of PACAP as a GH-releasing factor in fish, the structural identity of grass carp PACAP was established by molecular cloning. The newly cloned PACAP was found to be a single-copy gene and expressed in the brain but not in other tissues. The mature peptides of PACAP, namely PACAP₂₇ and PACAP₃₈, were synthesized. As revealed by NMR spectroscopies, carp PACAP₃₈ is composed of a flexible N-terminal from His¹ to Ile⁵, an extended central helix from Phe⁶ to Val²⁶, and a short helical tail in the C-terminal from Arg²⁹ to Arg³⁴. The C-terminal helix is located after a hinge region at Leu²⁷ to Gly²⁸ and is absent in the solution structures of PACAP₂₇. The two forms of PACAPs were effective in elevating GH release and GH transcript expression in grass carp pituitary cells. These stimulatory effects occurred with parallel rises in cAMP and Ca²⁺ entry via voltage-sensitive Ca²⁺ channels in carp somatotrophs. The present study represents the first report for solution structures of non-mammalian PACAPs and provides evidence that a brain-specific isoform of PACAP in fish can stimulate GH synthesis and release at the pituitary level, presumably by activating the appropriate post-receptor signaling mechanisms.