Isolated juvenile rainbow trout were fed a feed supplemented with L-tryptophan (TRP) for 3, 7 or 28 days, after which they were either sampled directly (undisturbed) or subjected to a standardised stressor prior to sampling. Controls (stressed and undisturbed) received the same feed but without any supplementary TRP. Stress resulted in a significant elevation of plasma [cortisol] in fish fed control feed and in fish fed TRP-supplemented feed for 3 and 28 days. However, fish fed TRP-supplemented feed for 7 days did not show any significant elevation of plasma [cortisol] in response to stress. Plasma levels of adrenocorticotropin followed the same general pattern as cortisol. Plasma and brain [TRP] were elevated in fish fed TRP-supplemented feed. The amino acid TRP is the precursor of the monoamine neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) and the brain 5-HT system is known to be involved in the control of the hypothalamic-pituitary-interrenal (HPI) axis. Fish fed TRP-supplemented feed showed elevated levels of 5-hydroxyindoleacetic acid (5-HIAA, a major 5-HT metabolite) in the hypothalamus and optic tectum. However, TRP treatment did not appear to result in any effects on brain dopaminergic activity and the effects on brain norepinephric activity do not support a role of norepinephrine in mediating the effects of TRP on HPI axis reactivity in rainbow trout.