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Melatonin is involved in the transduction of light information and the photoperiodic control of many important physiological functions in fish. Although artificial photoperiods have been used to improve fish growth and manipulate reproduction, there is little information about the characteristics of light ‘quality’. In this paper we describe the effects of a light pulse in the middle of the dark phase on plasma and ocular melatonin in European sea bass. We first determined the light intensity necessary to elicit a melatonin response using white light of varying intensities (0.6–600 μW/cm2, experiment 1). Secondly, we tested the effect of the light spectrum on melatonin production using three differently coloured lights (half-peak bandwidth = 434–477, 498–575 and 610–687 nm for the blue, green and red lamp, respectively, experiment 2) and, finally, we determined the effect of light orientation (downwards directed versus upwards directed, experiment 3). The results show that the minimum light intensity needed to inhibit or stimulate melatonin levels in both plasma and the eye was 6.0 μW/cm2. A linear correlation was found between the logarithm of light intensity and the relative inhibition. In addition, the blue wavelength was more effective in decreasing melatonin levels in the former and increasing the levels in the latter. Nevertheless, red light at sufficient intensity proved effective at significantly suppressing circulating melatonin. Downwards light had a greater effect than upward-directed illumination in suppressing plasma melatonin. In conclusion, the results point to the importance of giving proper consideration to the characteristics of light, to adequately control melatonin production and its related physiological processes.