The 35% CO(2) challenge is known to induce symptoms of a panic attack both in panic disorder (PD) patients and healthy volunteers. Although the challenge applies more to PD patients, studies in healthy volunteers provide the opportunity to isolate the physical symptoms from the disorder and to focus on the direct effect from the challenge on stress responsive systems. One of the main stress responsive systems is the hypothalamic-pituitary-adrenal (HPA) axis. It remains unclear whether panic symptoms are accompanied by HPA axis activation. Differences in design have hampered any comparison between studies. For example, both serum and salivary cortisol have been used to provide an index of HPA axis activation. Furthermore, indications for central HPA axis disturbance have been suggested. The current study aimed to study the HPA axis response following the induction of panic symptoms in healthy volunteers, both at the pituitary level and at the adrenal level. Furthermore, both serum and salivary cortisol levels were determined. Subjective feelings of anxiety and, correspondingly, cortisol and ACTH levels, were found to be significantly increased following the 35% CO(2) challenge. Cortisol and ACTH responses to CO(2) were also associated. A significant cortisol increase was observed in both serum and saliva samples, although these were more pronounced when considering the free fraction serum values. We conclude that the induction of panic symptoms results in HPA axis activation, both at the pituitary and adrenal level. The question remains as to whether positive responders to the 35% CO(2) inhalation (more specifically PD patients) show a more pronounced HPA axis response.