Objective Although stress has been considered an important pathophysiological factor in irritable bowel syndrome (IBS), there is incomplete understanding of its physiological mechanisms. The current study was designed to compare diurnal hypothalamic-pituitary-adrenal (HPA) axis activity in IBS patients and controls and their psychobiological response to a psychosocial stressor. Methods Basal and stimulated HPA axis activity was assessed in 57 women with IBS and 20 matched controls. Psychiatric comorbidity was assessed using a standardized clinical interview. Salivary morning cortisol and diurnal profile were obtained, and the Trier Social Stress Test (TSST) was administered. Levels of cortisol and adrenocorticotropic hormone (ACTH) were measured before and within 1 hour after the stressor. Overall stress experience and stress related to the TSST were assessed using standardized questionnaires. Results All subjects showed intact circadian variation of cortisol. However, IBS patients with predominant diarrhea exhibited substantially heightened cortisol levels at awakening (p < .03) and a blunted cortisol awakening response. In response to the TSST, patients exhibited significantly blunted cortisol (p < .05) and slightly attenuated ACTH secretion compared with controls. During the recovery period, ACTH levels were significantly lower (p < .04) in patients than those in healthy subjects. Women with IBS perceived higher stress susceptibility than control subjects did (p < .01). Conclusions The enhanced morning cortisol levels in one subgroup of IBS patients may indicate an association between basal HPA axis activity and predominant bowel habit. The downregulated HPA axis reactivity in IBS after the TSST suggests a downregulated sensitivity of the endocrine system. On the contrary, all subjective stress ratings were increased in the IBS group, which may indicate increased stress susceptibility.