According to current hypotheses, in mammals male-specific behavior is organized perinatally, and activated in adulthood by male gonadal hormones. However, this strict differentiation between early organizational and late activational hormone effects has been criticized recently. Around puberty the testosterone levels of male mammals rise far above adult levels. In this study we examined the relevance of this pubertal testosterone peak on marking behavior of adult tree shrews (Tupaia belangeri). Male tree shrews were castrated before puberty and treated with testosterone either around puberty or in adulthood. Several months later, the marking behavior of the castrated adult animals was examined in standard tests in different scent conditions before and after a second testosterone treatment. Males castrated as adults as well as fertile females were used as controls. The testosterone peak during puberty did not influence male marking behavior in the absence of scent from conspecifics (familiarization marking) or in the presence of female scent (sexual marking). However, testosterone during puberty determined both the male-specific reaction patterns to male scent (territorial marking) and the male-specific regulation of this behavior by testosterone. These results indicate that testosterone during puberty specifically organizes male territorial-marking behavior. To our knowledge, these are the first results demonstrating organizational effects of testosterone during puberty on male behavior.