The detailed distribution of neural nitric oxide synthase (nNOS)-positive cerebrospinal fluid-contacting neurons (CSF-CN) was studied in the wall of the third ventricle of rats by anti-nNOS immunohistochemistry. The coexistence of nNOS and 8-arginine vasopressin (AVP) or oxytocin (OT) was also investigated in the CSF-CN using double labeling immunohistochemistry. The results demonstrated a widespread occurrence of nNOS-CSF-CN throughout the wall of the hypothalamic third ventricle. The vast majority of nNOS-CSF-CN cell bodies were of magnocellular type, commonly classified as oval, fusiform, multipolar, and inverted pear shape. These cell bodies were located in the ependyma, the subependyma, or the parenchyma, and their processes inserted in the ependymal layer or directly contacted with the CSF space. Electron microscopy demonstrated many nNOS-immunoreactive somas, dendrites, and/or axons that were situated at the subependyma, the ependyma, or the supraependyma. Generally, the distribution of OT-CSF-CN in the third ventricular wall was similar to the nNOS-CSF-CN and the ratio of NOS/OT co-expression was approximately 88%. In comparison, the distribution of AVP-CSF-CN was mainly restricted to the rostral part of the third ventricle and the ratio of nNOS/AVP co-expression was only about 6%. The widespread presence of nNOS-CSF-CN-expressing OT in the third ventricular region suggests that NO is an important messenger in the CSF-hypothalamo-hypophyseal neuroendocrine regulation that may in part act in concert with OT.