It has been shown that cortisol concentrations change characteristically in the course of agonistic interactions; our aim was to find out how a playful situation may affect concentrations of this hormone in the saliva. We studied dogs' behavior and the changes of cortisol concentrations in a play situation, where the dogs played with their handler for 3 min with a tug toy. In this experiment working dogs were divided into two groups by the type of their work, namely police dogs and border guard dogs. We found that the cortisol concentrations of old police dogs significantly increased, while the adult border guard dogs' hormone levels decreased, which shows that playing with the handler has an effect on both groups, but interestingly this effect was opposite. Behavior analysis showed differences only in the behavior of the human handlers during the play sessions, while the behavior analysis did not reveal significant differences in the two groups of dogs, except that old border guard dogs generally needed more time to begin playing than old police dogs. During the play sessions police officers were mainly disciplining their dogs, while the border guards were truly playing with them (including affiliative and affectionate behavior). Our results are in accordance with those of recent studies, which show that behaviors associated with control, authority or aggression increase cortisol concentrations, while play and affiliative behavior decrease cortisol levels.