Problematic gambling is thought to be influenced by neurobiological mechanisms. However, the neuroendocrine response to gambling is largely unknown. Therefore, the effect of casino gambling on the sympathoadrenal system, the HPA-axis, and pituitary hormones were analyzed. Fourteen male problem gamblers and 15 non-problem gamblers were examined in a balanced cross-over design. In the experimental session, participants played blackjack in a casino wagering their own money. During the control session, subjects played cards for accumulation of points. Heart rate and endocrine measures were recorded at baseline, at 30, 60 and 90 min during gambling/card playing, and after the game. Heart rate and norepinephrine levels increased with the onset of blackjack in both groups, with problem gamblers showing significantly higher levels across the entire gambling session. In addition, dopamine levels were significantly higher in problem gamblers during casino gambling compared to non-problem gamblers. Cortisol levels were transiently increased with the onset of blackjack in both groups. Casino gambling as a "real life" situation induces activation of the HPA-axis and the sympathoadrenergic system, with significantly more pronounced changes in problem gamblers. These findings may contribute to a better understanding of neuroendocrine disturbances in problem gambling.