Bioluminescence signal imaging is a powerful tool to visualize gene expression in not only living cells but also living animals. We previously generated a novel transgenic mouse strain, termed BDNF-Luc Tg mouse, to monitor BDNF gene expression using luciferase as an imaging probe. Also, we have already found that a neuropeptide, PACAP, increased the mRNA level for BDNF via NMDA receptor-calcineurin pathway. In this study, we further investigated how PACAP induces BDNF gene expression in neurons, using a primary culture of cortical cells prepared from BDNF-Luc Tg mouse embryos. Although PACAP increased BDNF gene expression at 5 days in culture, the increase was observed in a few cells. On the other hand, the robust increase in BDNF gene expression was observed in many cells when PACAP was added into the cells at 14 days in culture. The increase in BDNF gene expression at 5 and 14 days in culture was canceled by the pretreatment with APV or FK506, an antagonist of NMDA receptor and an inhibitor for calcineurin, respectively, corresponding to the change in endogenous BDNF mRNA expression. Since spontaneous calcium oscillation, which was mediated at least in part by NMDA receptor, was observed in mature cortical cells (14 DIV), and hardly in immature cells (5 DIV), we hypothesized that PACAP selectively activates NMDA receptor-calcineurin pathway, which operates by endogenous glutamatergic neurotransmission in culture. To test this hypothesis, we exogenously added NMDA, an agonist of NMDA receptor, to directly activate NMDA receptor in immature cells, in which endogenous glutamatergic neurotransmission rarely occurs. As compared with PACAP, NMDA increased BDNF gene expression in many cells. Interestingly, PACAP synergistically enhanced the NMDA-induced BDNF gene expression. This synergistic induction was completely inhibited by FK506. These results indicate that PACAP selectively activates calcineurin pathway via NMDA receptor to effectively induce BDNF gene expression in neurons.