Transcription of arylalkylamine N-acetyltransferase 2 (aanat2) gene leads to the formation of AANAT2 - the rate-limiting enzyme in melatonin synthesis in the photosensitive fish pineal. Unlike in mammalian pineal, there is practically no information on signal transduction pathway(s) involved in the regulation of aanat2 gene transcription in the fish pineal. Therefore, we investigated the role of important molecular components of signalling via cAMP, cGMP, and Ca2+ involving cAMPdependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), Ca2+-dependent protein kinase (PKC), mitogen-activated protein kinase (MAPK) kinase (MEK) and p38 MAPK (p38 MAPK) as well as the possible role of serine/threonine phosphatases (PPs), cAMP response element binding protein (CREB) and CREB binding protein (CBP) using their specific inhibitors in regulation of aanat2 transcripts in the fish pineal under in vitro conditions. db-cAMP and db-cGMP increased AANAT2 mRNA levels. db-cAMP- and db-cGMP-induced increase in AANAT2 mRNA levels was significantly reduced in the presence of H-89 (PKA inhibitor), KT5823 (PKG inhibitor), chelerythrine chloride (PKC inhibitor), U0126 (MEK inhibitor), and SB 202190 (p38 MAP kinase inhibitor). Inhibitors of PP1 and PP2A significantly increased AANAT2 mRNA as well as significantly reduced db-cAMP and db-cGMP induced increase in AANAT2 mRNA levels. Inhibitors of both CREB and CBPCREB interaction completely blocked the cAMP-induced increase in AANAT2 mRNA levels. Based on these findings, we suggest that cAMP, cGMP, and Ca2+ increase AANAT2 mRNA levels via PKA, PKG, and PKC, respectively. Further, protein phosphatases and the CBP-CREB-CRE pathway are actively involved in the regulation of AANAT2 mRNA levels in the fish pineal.