Borderline personality disorder (BPD) is characterized by a pervasive pattern of instability in affect regulation, impulse control, interpersonal relationships, and self-image. In previous studies, we have used portable mini-computers to assess the severity of recurrent states of aversive emotional distress and dissociation during ambulatory conditions. Here, we used this approach for the assessment of the hypothalamic-pituitary-adrenal (HPA) axis in patients with BPD. We studied 23 unmedicated female patients with BPD and 24 matched healthy controls. Salivary cortisol was collected from all participants during ambulatory conditions in response to reminders provided by portable mini-computers on 3 consecutive days every 2 h for 14 h after awakening. In addition, cortisol in response to awakening was determined in four 15 min intervals on days 1 and 2. After the last collection of cortisol on the second day, 0.5 mg dexamethasone was administered in order to achieve cortisol suppression on day 3 (low-dose dexamethasone suppression test, DST). Patients with BPD displayed significantly higher salivary cortisol levels than healthy controls as demonstrated by higher total cortisol in response to awakening and higher total daily cortisol levels. There were significantly more non-suppressors of cortisol in the low-dose DST in the patient group when compared to the control group. The ambulatory assessment of saliva cortisol is a suitable approach to study basic parameters of the HPA-axis in patients with BPD. Increased adrenal activity and lowered feedback sensitivity of the HPA-axis may characterise BPD. Further studies have to reveal reasons of heightened adrenal activity in these patients.