A dysfunctional hypothalamic pituitary adrenal (HPA) axis is widely accepted as a significant pathophysiological aspect of Major Depressive Disorder (MDD). Despite studies suggesting that a dysfunctional HPA axis might be present before the clinical syndrome becomes apparent, the functioning of the HPA axis in high-risk populations has not been well defined. The aim of the present study was to investigate the HPA axis functioning of mothers suffering from MDD and their healthy daughters compared to age- and sex-matched healthy controls. This design allowed a comparison of HPA axis functional differences among daughter and mother groups. HPA axis function was evaluated with a modified dexamethasone/corticotropin-releasing hormone (Dex/CRH) test, which was performed after obtaining the diurnal adrenocorticotropic hormone (ACTH) and cortisol values at 8:00, 16:00, and 23:00 h. We found that MDD mothers and their daughters had low morning cortisol and the MDD mothers additionally had low-morning ACTH compared with controls. Dexamethasone suppressed both cortisol and ACTH in all groups and subsequent HPA axis stimulation by CRH-evoked a lower cortisol response but a higher ACTH response among subjects with MDD mothers. Although high-risk daughters had comparable cortisol levels after CRH infusion, the AUC for ACTH was greater than those of controls. These patterns of results suggest that multiple level HPA dysfunctions are present in both MDD patients and their high-risk carrying daughters. However, insufficient cortisol secretion was only present in MDD mothers, while the daughters could compensate cortisol levels during CRH challenge.