Rough and tumble (R&T) play is an intrinsic behavior in most mammals. However, unlike sex and aggression, play has not been well characterized in terms of neuronal circuitry. We employed in situ hybridization to explore the differences of c-fos mRNA activation in juvenile rats that had been allowed R&T play for a total of 30 min before sacrifice contrasted to animals with comparable histories that had received no play. Densitometric estimates of c-fos gene activation revealed that the deep and dorsolateral tectum, inferior colliculus, dorsal periaquaductal gray, ventromedial hypothalamus, dorsal and ventral striatum, and somatosensory cortex were significantly more activated in animals that had played than those that had not. Prior play dominance and amount of social experience had no clear effects on the levels of c-fos gene expression. This provides a variety of new hypotheses concerning the role of various brain areas in the elaboration of R&T play behavior, but the important role of other types of motor arousal in the differential effects were not evaluated in this study.