The present experiments were designed to test the hypothesis that elevated dietary levels of l-tryptophan (Trp) result in elevated plasma levels of melatonin and that this increase in plasma melatonin concentration is caused by elevated melatonin production and secretion by the gastro-intestinal-tract (GIT). Feeding juvenile rainbow trout (Oncorhynchus mykiss) Trp-supplemented feed for 7 days resulted in elevated daytime plasma levels of melatonin and reduced poststress plasma cortisol concentrations. Nighttime plasma melatonin concentrations were, however, not affected by elevated dietary Trp. Moreover, stress caused a reduction in daytime plasma levels of melatonin in fish fed Trp-supplemented feed, an effect that was counteracted by treatment with an alpha-receptor antagonist. These results clearly suggest that elevated dietary intake of Trp results in an increase in the GIT production of melatonin in rainbow trout. A suggestion that was further supported by the results from an in vitro experiment demonstrating that addition of Trp to the incubation medium stimulates melatonin production and release by incubated rainbow trout GIT. The results from this study led us to suggest a possible mechanism for melatonin in mediating the effects of elevated dietary Trp on poststress plasma cortisol concentrations and aggressive behavior in rainbow trout.