Social play behavior is one of the earliest forms of non-mother-directed social behavior appearing in ontogeny in mammalian species. During the last century, there has been a lot of debate on the significance of social play behavior, but behavioral studies have indicated that social play behavior is a separate and relevant category of behavior. The present review provides a comprehensive survey of studies on the neurobiology of social play behavior. Evidence is presented that opioid and dopamine systems play a role in the reward aspect of social play behavior. The role of cholinergic, noradrenergic and opioid systems in attentional processes underlying the generation of social play behavior and the involvement of androgens in the sexual differentiation of social play behavior in rats is summarized. It is concluded that there is not only behavioral, but also neurobiological evidence to suggest that social play behavior represents a separate category of behavior, instead of a precursor for adult social, sexual or aggressive behavior.