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The pineal gland of the zebrafish (Danio rerio) is a clock-containing photoreceptive organ. Superfused pineal glands kept in darkness display rhythmic melatonin production that lasts for days, with high melatonin levels during the night and low levels during the day. Nocturnal light, however, evokes an acute suppression of melatonin synthesis in the photoreceptor cells. Towards characterizing zebrafish pineal photopigment that is involved in the acute melatonin suppression we have measured the spectral sensitivity of melatonin-suppression response in superfused pineal glands. The effect of 2 h light exposure of seven wavelengths (λavg 408, 460, 512, 560, 608, 660 and 697±10–15 nm) at multiple irradiances (107–1014 photons/cm2/s) was determined, and an action spectrum was plotted. The resultant action spectrum provides evidence for the involvement of multiple photopigments in melatonin suppression. The most efficient melatonin-suppression response was achieved by exposure to light of around 512 nm; however, another peak of lower irradiance sensitivity was observed in the middle to long wavelengths. Opsins-specific RT-PCR analysis confirmed the expression of exo-rhodopsin and visual red-sensitive opsin in the pineal gland, while other zebrafish visual opsins as well as VA and VAL opsins were not detected. Dartnall monograms for exo-rhodopsin and visual red-sensitive opsin account for most but not all of the spectral sensitivity features. Therefore, additional pineal photopigments may contribute to the melatonin-suppression response in the pineal gland.