Background: Children of parents with a substance use disorder (CPSUDs) are at increased risk for the development of substance use disorders later in life, and therefore may manifest vulnerability markers for these disorders at a higher level than children from the general population. Our aim was to examine hypothalamic-pituitary-adrenal (HPA) axis activity as a potential vulnerability marker in CPSUDs as compared to healthy controls. We further examined whether having experienced more adverse life events (ALEs) accounted for differences in cortisol levels between CPSUDs and controls. Methods: 83 CPSUDs were matched to 83 controls on the basis of age, sex and socioeconomic status. Salivary cortisol was assessed at four time points during a normal day and at six time points during a psychosocial stress procedure, during which perceived stress was also measured. We implemented piecewise multilevel growth curve modeling to examine group differences in diurnal and stress-evoked cortisol levels. Results: Diurnal cortisol levels of CPSUDs did not differ from those of controls. Only stress-evoked cortisol levels at onset of the experiment were explained by group status, such that CPSUDs exhibited lower cortisol levels at onset of the stress procedure. CPSUDs reported experiencing significantly more ALEs, yet number of ALEs was not related to cortisol levels. CPSUDs furthermore reported less perceived stress than controls at onset of the procedure. Conclusions: HPA axis dysregulation may be a vulnerability marker for substance use disorders, as CPSUDs show blunted activation in anticipation of stress. These blunted cortisol levels were not the result of having experienced more stressful experiences during their lifetimes, thus might reflect an inborn vulnerability to substance use disorders. © 2013 Elsevier Ltd.