Arginine vasopressin (AVP) is a neuropeptide involved in the regulation of fluid balance, stress, circadian rhythms, and social behaviors. In the brain, AVP is tightly regulated by gonadal steroid hormones in discrete regions with gonadectomy abolishing, and testosterone (T) replacement restoring normal AVP expression in adult males. Previous studies demonstrated that 17-estradiol (E2), a primary metabolite of T, is responsible for restoring most of the AVP expression in the brain following castration. However, 5-dihydrotestosterone (DHT) has also been shown to play a role in the regulation of AVP expression, thus implicating the involvement of both androgen (AR) and estrogen receptors (ER). Further, DHT, through its conversion to 3Adiol, has been shown to modulate ERE-mediated promoter activity through an estrogen receptor pathway. The present study addressed two central hypotheses: 1) that androgens directly modulate AVP promoter activity and 2) the effect is mediated by an estrogen or androgen receptor pathway. To that end, we over-expressed AR, ER, and ER splice variants in a neuronal cell line and measured AVP promoter activity using a firefly luciferase reporter assay. Our results demonstrate that DHT and its metabolite 3Adiol stimulate AVP promoter activity through estrogen receptor-beta (ER) in a neuronal cell line.