Ghrelin (GRLN) and its receptor have been identified and characterised in goldfish brain and the pituitary, and recent evidence shows that goldfish (g)GRLN19 induces both growth hormone (GH) and maturational gonadotrophin (LH) release through an extracellular Ca2+-dependent mechanism in goldfish. To further understand the role of GRLN in hormone release, the present study examined the involvement of protein kinase C (PKC) and protein kinase A (PKA) in gGRLN19-induced GH and LH release and corresponding Ca2+ signals in primary cultures of goldfish pituitary cells. Treatments with PKC inhibitors, Bis-II and Gö 6976, significantly reduced gGRLN19-induced GH and LH release and their corresponding intracellular Ca2+ signals in identified somatotrophs and gonadotrophs, respectively. gGRLN19 was unable to further stimulate hormone release or Ca2+ signals when cells were pretreated with the PKC agonist, DiC8. PKA inhibitors, H-89 and KT 5720, inhibited gGRLN19-induced LH release and Ca2+ signals in gonadotrophs but not GH release or Ca2+ signals in somatotrophs. Interestingly, pretreatment of pituitary cells with the adenylate cyclase activator forskolin potentiated gGRLN19-induced GH, but not LH, release, although it had no effect on intracellular Ca2+ signals in either cell type. Taken together, the results suggest that PKC is an important intracellular component in gGRLN19-induced GH and LH release, whereas PKA is involved in gGRLN19-elicited LH release. Furthermore, the PKA pathway potentiates gGRLN19-induced GH release via a Ca2+-independent mechanism. Overall, the present study provides insight into the neuroendocrine regulation of GH and LH release by elucidating the mechanistic aspects of GRLN, a hormone involved in many critical physiological processes, including pituitary functions.