Defining how arginine vasopressin (AVP) acts centrally to regulate homeostasis and behavior is problematic, as AVP is made in multiple nuclei in the hypothalamus (i.e., paraventricular [PVN], supraoptic [SON], and suprachiasmatic [SCN]) and extended amygdala (i.e., bed nucleus of the stria terminalis [BNST] and medial amygdala [MeA]), and these groups of neurons have extensive projections throughout the brain. To understand the function of AVP, it is essential to know the site of origin of various projections. In mice, we used gonadectomy to eliminate gonadal steroid hormone– dependent expression of AVP in the BNST and MeA and electrolytic lesions to eliminate the SCN, effectively eliminating those AVP- immunoreactive projections; we also quantified AVP- immunoreactive fiber density in gonadectomized and sham- operated male and female mice to examine sex differences in AVP innervation. Our results suggest that the BNST/MeA AVP system innervates regions containing major modulatory neurotransmitters (e.g., serotonin and dopamine) and thus may be involved in regulating behavioral state. Furthermore, this system may be biased toward the regulation of male behavior, given the numerous regions in which males have a denser AVP- immunoreactive innervation than females. AVP from the SCN is found in regions important for the regulation of hormone output and behavior. Innervation from the PVN and SON is found in brain regions that likely work in concert with the well- known peripheral AVP actions of controlling homeostasis and stress response; female- biased sex differences in this system may be related to the heightened stress response observed in females. J. Comp. Neurol. 521:2321– 2358, 2013. © 2012 Wiley Periodicals, Inc.