Acute tryptophan depletion (ATD) is a technique that has been used to evaluate the effects on humans of acutely reducing serotonin neurotransmission. We have developed a model using a single breath of 35% CO2 that activates the hormonal axis and produces autonomic and behavioural arousal, thus modelling a stress response. This study combines ATD and single breath 35% CO2 inhalation to study stress responses in volunteers.A randomised, double-blinded, placebo-control Led, cross-over trial involving 14 healthy adult volunteers aged between 18 and 65 years was undertaken. Subjects underwent double-blind tryptophan depletion over 2 days and were then crossed over 1 week later. During each study day, at the time of peak depletion, participants were single blinded to receive a single breath of 35% CO2 or air. This was followed 40 min later by the other gas. Psychological outcomes were assessed with the Spielberger State Anxiety Inventory (SSAI), Visual Analogue Scales (VAS), Panic Inventory (PI), Panic and Agoraphobia Scale (PSI) and Beck Depression Inventory (BDI). Physiological outcome was measured by serial plasma cortisol, prolactin and tryptophan levels, pulse and blood pressure.Tryptophan depletion did not exacerbate 35% CO2 inhalation effects on anxiety symptoms. Single breath CO2 robustly increased plasma cortisol Levels in comparison to an air inhalation; this was less certain for prolactin Levels. ATD influenced the HPA axis (associated with higher cortisol levels), apparently independent Of CO2 or air inhalation stressors. ATD and 35% CO2 inhalation both induced a pressor response and bradycardia in these normal volunteers.Thirty-five percent CO2 inhalation and ATD independently activate the human stress response, but do not appear to produce synergistic effects when combined, at least for the conditions produced in this study. (c) 2006 Elsevier Ltd. All rights reserved.