Play behavior was assessed in two inbred strains of rats. Rats of the Fischer-344 strain (F344) were found to be less playful than Buffalo rats after social isolation, as evident from fewer pins and fewer playful attacks to the nape. When tested in same-strain pairings, overall defense of the nape did not differ between strains, although there were strain differences in the specific pattern of defense. When tested in cross-strain pairings, F344 rats were less likely to direct nape attacks toward a Buffalo play partner, and were also less likely to defend their nape when attacked by Buffalo rats. Although different levels of pinning and nape attacks in the two strains were dependent on the amount of isolation prior to the play period, differences in nape defense were not dependent on prior isolation. This pattern of results suggests that the neural mechanisms for playful attack differ from those underlying playful defense. These data also suggest that the F344 strain could be useful in better understanding the neural and genetic bases of mammalian playfulness.