Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. However, the signal transduction mechanisms responsible for SL expression are still largely unknown. Using grass carp as an animal model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL gene expression at the pituitary level. In primary cultures of grass carp pituitary cells, SL and SL mRNA levels could be elevated by PACAP via activation of PAC-I receptors. Using a pharmacological approach, the AC/cAMP/PKA and PLC/IP3/PKC pathways and subsequent activation of the Ca²⁺/CaM/CaMK-II cascades were shown to be involved in PACAP-induced SL mRNA expression. Apparently, the downstream Ca²⁺/CaM-dependent cascades were triggered by extracellular Ca²⁺ ([Ca²⁺]e) entry via L-type voltage-sensitive Ca²⁺ channels (VSCC) and Ca²⁺ release from IP3-sensitive intracellular Ca²⁺ stores. In addition, the VSCC component could be activated by cAMP/PKA- and PLC/PKC-dependent mechanisms. Similar post-receptor signaling cascades were also observed for PACAP-induced SL mRNA expression, except that [Ca²⁺]e entry through VSCC, PKC coupling to PLC, and subsequent activation of CaMK-II were not involved. These findings, taken together, provide evidence for the first time that PACAP can induce SL and SL gene expression in fish model via PAC-I receptors through differential coupling to overlapping and yet distinct signaling pathways.