In the present study, anterograde tracing combined with triple label immunofluorescent staining was conducted to examine the possible anatomical interactions between Neuropeptide Y (NPY) neurons in the arcuate nucleus of the hypothalamus (ARH) and the corticotropin releasing hormone (CRH) system in the paraventricular nucleus of the hypothalamus (PVH). The anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), was iontophresed into the ARH of female rats and triple label immunofluorescence staining with three different fluorophores was performed to visualize PHA-L, NPY and CRH, with the aid of confocal microscopy. In PVH, NPY and PHA-L double-labeled fibers were found mainly in the parvocellular part of the PVH (PVHp). Confocal analysis demonstrated that NPY/PHA-L double-labeled fibers came in close apposition to CRH perikarya. In the median eminence, NPY/PHA-L double-labeled fibers were found both in the inner and the outer zones of the median eminence. However, very few double-labeled fibers were found in the proximity of CRH neuronal fibers in the median eminence. Double label staining was also performed to determine if NPY Y1 receptors were expressed in CRH neurons. Two different fluorophores were used to visualize CRH neurons and Y1 receptor. No convincing Y1-positive staining was found in CRH cell bodies in the PVH, even though Y1-positive staining in numerous fibers and cell bodies was observed throughout the region. However, Y1-positive fibers were shown to make close contact with CRH cell bodies in the PVH. In the ME, the majority of the Y1-positive fibers were located in the lateral portion of the ME, whereas the CRH fibers were found mainly in the medial portion of the external zone of the ME. The results of the present study suggest that ARH NPY neurons provide direct input into CRH cell bodies in the PVH region. However, the direct effects of NPY must be mediated by some receptor subtype other than Y1. Y1 receptor involvement in NPY modulation of CRH neuronal function in the PVH appears to be indirect through modulation of neuronal afferents making contact with CRH neurons.