The hypothalamic–pituitary–adrenal axis (HPA axis) plays a critical role in stress management. Involvement of this physiological axis in the underlying mechanisms of panic disorder (PD) has been suggested. Studies using 35% CO2 inhalation to provoke panic found no evidence for robust increases in cortisol levels in PD. However, cortisol levels alone may not be conclusive, as this hormone is merely the end product of a complex physiological axis. Sixteen PD patients and 16 healthy control subjects underwent a 35% CO2 inhalation and a placebo inhalation on separate days according to a fixed order, double-blind design. Both serum and salivary cortisol, as well as adrenocorticotropic hormone (ACTH) were measured at regular time intervals. Cortisol and ACTH levels increased in the patient and control groups following 35% CO2 inhalation. The magnitude of the increase was similar in patients and controls despite marked differences in anxiety. This study is the first to document a clear HPA response following 35% CO2 inhalation in both PD patients and controls. This effect occurs independently of the specific panicogenic properties of the CO2 challenge. It remains to be clarified whether panic is initially accompanied by major HPA axis activation or whether other stress-responsive systems underlie panic.