Introduction: Tryptophan depletion (TD) is an established method to influence the serotonergic system and mood. The purpose of this study was to examine the effect of TD under different ambient light conditions, measured through serotonin-associated plasma levels and a visual analog scale (VAS), on healthy females. Methods: Thirty-eight healthy female s-allele carriers of the serotonin transporter promoter gene (5-HTTLPR) were administered a TD under dim light conditions (75 lx). A sub-group of 8 participants repeated the procedure randomized in two additional light conditions (585 lx and 1530 lx respectively). Prior to, and 5 h following administration of TD, various variables (serotonin-associated plasma levels, VAS) were measured. Due to not normal distributed data, non-parametric statistical tests were used. Results: Overall analysis showed a significant mood lowering effect of TD. Moreover, TD decreased all measured serotonin-associated plasma levels significantly. Significant differences in varying light conditions were found for the VAS and plasma tryptophan, with the greatest effect of TD in the 75 lx condition. Conclusion: Results of our study showed an influence of even slight differences in ambient light intensity on the effect of TD concerning mood as well as on the serotonergic system. (C) 2013 Elsevier Ireland Ltd. All rights reserved.