Juvenile rats engage in rough-and-tumble play, and these interactions are important for the development of adult social behaviors. Recently, we examined Fos expression in dopaminergic neurons of the ventral tegmental area (VTA) after juvenile rats played or did not play in an undisturbed paradigm1 . Play was associated with more Fos expression in VTA dopaminergic neurons in females, but this effect was not observed in males. It is possible that brief play durations in males (139 s in a one-hour observation) may not have been sufficient to elicit Fos expression in VTA dopaminergic neurons. To determine whether longer play times would increase Fos expression in males, we separated subjects from their siblings at weaning so that subjects could be tested with a same-sex sibling that they had not interacted with in 9-11 days. After testing with the “unfamiliar” sibling or isolation (control), subjects were perfused, immunohistochemistry for both tyrosine hydroxylase and Fos was performed, and the number of double-labeled cells in the VTA was counted. Interestingly, separating siblings for 1.5 weeks did not increase play duration, but the number of double-labeled cells in the VTA was higher in both males and females playing with an “unfamiliar” sibling than in controls. Furthermore, our data suggest that there may be even greater Fos expression in males that have played than in females that have played in this paradigm. We are further exploring whether early postnatal actions of gonadal hormones are important for masculinizing this neural response, and preliminary data suggest that daily injections of an aromatase inhibitor and flutamide on postnatal days 1-7 do not influence juvenile play-induced Fos expression in males. Our results indicate that dopamine release from mesolimbic dopaminergic neurons during play may depend on the familiarity of the play partner, particularly for males.