Background: Heroin dependence is a chronic relapsing disorder characterized by a compulsion to seek and use heroin despite the negative consequences. In addition to the diagnostic criteria, heroin dependence is associated with an altered function of the hypothalamic-pituitary-adrenal (HPA) axis and with affective disturbances and other mental disorders. Stress and heroin craving are considered as key motivators for heroin use. Heroin-assisted treatment (HAT) is suitable for chronic heroin-dependent patients who do not benefit of other therapy forms. Methods: The acute effects of intravenous diacetylmorphine (DAM, pharmaceutical heroin) on the HPA axis response and on the emotions in heroin-dependent patients compared with placebo and with healthy controls were examined. Twenty-eight heroin-dependent patients in stable HAT and twenty age and gender matched healthy controls were recruited for a randomized, controlled crossover trial. Patients were administered heroin (DAM) or placebo (saline), healthy controls received only saline. HPA axis response was measured by adrenocorticotropic hormone (ACTH) and by cortisol concentrations in plasma, serum and saliva to three time points (before, 20 and 60 minutes after the substance administration). Withdrawal syndrome, craving, mood, anxiety and anger were measured before and 60 minutes after the substance application using validated questionnaires. Plasma concentrations of heroin and its main metabolites were assessed using high-performance liquid chromatography. Results: Compared to placebo, DAM administration reduced withdrawal, anxiety and anger in heroin-dependent patients and was associated with significant decreases in the ACTH and cortisol concentrations (p < 0.01). After placebo, all hormone levels were significantly higher in patients than in healthy controls (p < 0.01). When patients received DAM, their cortisol concentrations did not differ from healthy controls and their ACTH levels were significantly lower (p < 0.01). The concurrent cocaine use had no significant influence on the HPA axis response. Before substance injection, heroin-dependent patients showed significantly higher anxiety and depression scores than healthy controls (p < 0.0001). Irrespective of the patients’ perceived intoxication and sedation, DAM administration was associated with a significant decrease in all negative emotions and in heroin craving, and with a significant increase in emotional well-being (p < 0.0001). When patients received DAM, they did not differ from healthy controls in their emotions at the end of the experiment. Conclusion: Acute DAM administration suppresses the stress response, dampens craving and negative emotions and increases positive emotions in heroin-dependent patients in a stable opioid maintenance program (HAT). Patients showed normalized HPA axis responses and emotional states, when they received their regular DAM dose. These findings indicate that heroin (DAM) - considered as a stress-protective factor – contributes to the regulation of the stress sensitivity and emotional vulnerability in heroin-dependent patients, and thus underscore the clinical benefit of HAT for chronic heroin dependence. Key-words: Heroin dependence; Adrenocorticotropic hormone (ACTH); Cortisol; Diacetylmorphine (DAM); Heroin-assisted treatment (HAT); Hypothalamic-pituitary-adrenal (HPA) axis; Craving; Negative emotions