Abstract The hormone melatonin connects environmental cues, such as photoperiod and temperature, with a number of physiological and behavioural processes, including seasonal reproduction, through binding to their cognate receptors. This study reports the structural, functional and physiological characterization of five high-affinity melatonin receptors (Mtnr1aaα, Mtnr1aa?, Mtnr1ab, Mtnr1al, Mtnr1b) in Atlantic salmon. Phylogenetic analysis clustered salmon melatonin receptors into three monophyletic groups, Mtnr1A, Mtnr1Al and Mtnr1B, but no functional representative of the Mtnr1C group. Contrary to previous studies in vertebrates, pharmacological characterization of four receptors in COS-7, CHO and SH-SY5Y cell lines (Mtnr1Aaα, Mtnr1Aa?, Mtnr1Ab, Mtnr1B) showed induction of intracellular cAMP levels following 2-iodomelatonin or melatonin exposure. No consistent response was measured after N-acetyl-serotonin or serotonin exposure. Melatonin receptor genes were expressed at all levels of the hypothalamo-pituitary-gonad axis, with three genes (mtnr1aa?, mtnr1ab and mtnr1b) detected in the pituitary. Pituitary receptors displayed daily fluctuations in mRNA levels during spring, prior to the onset of gonadal maturation, but not in autumn, strongly implying a direct involvement of melatonin in seasonal processes regulated by the pituitary. To the best of our knowledge, this is the first report of cAMP induction mediated via melatonin receptors in a teleost species.