The hypothalamic neuropeptide Y (NPY) circuitry is a key regulator of feeding behavior. NPY also acts in the mesolimbic dopaminergic circuitry, where it can increase motivational aspects of feeding behavior through effects on dopamine output in the nucleus accumbens (NAc) and on neurotransmission in the ventral tegmental area (VTA). Endogenous NPY in the NAc originates from local interneurons and afferent projections from the hypothalamic arcuate nucleus (Arc). However, the origin of endogenous NPY in the VTA is unknown. We determined, in normal-weight male Wistar rats, if the source of VTA NPY is local, and/or whether it is derived from VTA-projecting neurons. Immunocytochemistry, in situ hybridization and RT-qPCR were utilized, when appropriate in combination with colchicine treatment or 24h fasting, to assess NPY/Npy expression locally in the VTA. Retrograde tracing using cholera toxin beta (CTB) in the VTA, fluorescent immunocytochemistry and confocal microscopy were used to determine NPY-immunoreactive afferents to the VTA. NPY in the VTA was observed in fibers, but not following colchicine pre-treatment. No NPY- or Npy-expressing cell bodies were observed in the VTA. Fasting for 24h, which increased Npy expression in the Arc, failed to induce Npy expression in the VTA. Double-labeling with CTB and NPY was observed in the Arc and in the ventrolateral medulla. Thus, VTA NPY originates from the hypothalamic Arc and the ventrolateral medulla of the brainstem in normal-weight male Wistar rats. These afferent connections link hypothalamic and brainstem processing of physiologic state to VTA-driven motivational behavior. This article is protected by copyright. All rights reserved.