Testosterone (T) is known to play a central role in the facilitation of male-type social behavior, such as sexual and aggressive, and the development of their neural bases in male mice. Moreover, the action of testosterone via estrogen receptor (ER) ɑ, after being aromatized to estradiol in the brain, is suggested to be crucial for the full expression of these behaviors. We previously reported that knocking down of ERɑ with the use of virally mediated RNAi method in the medial preoptic area (MPOA) in adult male mice greatly reduced sexual without affecting aggressive behavior, while that in the medial amygdala (MeA) had no effect on either behavior (Sano et al. EJN, 2013). Recent studies have shown that T stimulation in pubertal period may have a critical role for full expression of male social behavior in adulthood. However, it is still not known whether and in which brain region ERɑ is involved in this developmental effect of T. In this study, we examined the effects of site-specific knockdown of ERɑ during pre-pubertal period. At the age of 21 days, gonadally intact male mice (ICR/Jcl) were bilaterally injected either with adeno-associated viral vector silencing ERɑ or a control vector in the MeA or MPOA. All mice were then tested for their sexual and aggressive behaviors starting at 12 weeks old. We found that pre-pubertal knockdown of ERɑ in the MeA reduced both sexual and aggressive behaviors while that in the MPOA only reduced sexual, but not aggressive behavior. Furthermore, number of MeA neurons examined in adult was reduced by pre-pubertal knockdown of ERɑ. These results suggest that although it may not be required at the time of testing, ERɑ activation in the MeA during pubertal period is crucial for male mice to fully express their male-type social behaviors later in adulthood. (Supported by KAKEN #23240057 to SO.)