In a previous study we found that a tryptophan-deficient amino acid mixture, designed to lower tissue tryptophan and thus brain 5-hydroxytryptamine (5HT) levels, caused a rapid (5 h) lowering of mood in normal males. Because of the importance of this evidence indicating a direct causal connection between low 5HT and low mood, we have now investigated other possible explanations for the mood lowering effect. Research strongly supports the involvement of environmental setting and cognition in the production and experience of emotions. Therefore we investigated how these factors might influence the mood-lowering effects of tryptophan depletion. In an instructional manipulation subjects were either supplied or not supplied with information designed to account for any possible peripheral sensations that might be related to depressive affect. In an environmental manipulation subjects were exposed either to a supportive and comfortable atmosphere (positive environment), or an unrewarding and unstimulating environment (negative environment). In the control group, which received a balanced amino acid mixture, the positive and negative environments had the expected effects on the scores of the Multiple Affect Adjective Checklist, thus indicating the effectiveness of these procedures. In the tryptophan depletion group neither the instructional nor the environmental manipulation had any influence on the mood lowering effect. It may be that tryptophan depletion lowers mood in normal males because low 5HT influences mood directly rather than via cognitive processes. Our data strongly support the idea that 5HT exerts an effect on mood and that low 5HT may, in some patients, be an important factor contributing to the etiology of clinical depression.