Central to this investigation are several basic hypotheses that are designed to test the role of nitric oxide (NO) in the complex process of central regeneration and plasticity in a well established model system of the mammalian brain. We have employed histochemical techniques at the light and ultrastructural level coupled with correlative scanning electron microscopy, immunoelectron microscopy, and in situ hybridization in order to determine the functional significance of the increased expression of nitric oxide synthase (NOS) in neurons of the supraoptic (SON) and paraventricular (PVN) nuclei which accompanies regeneration of their axotomized neurites following hypophysectomy. The aim of this investigation was to determine the potential role and temporal up-regulation of NOS in this basic regenerative process and to establish the ultrastructural and neuroanatomical correlates during critical periods of regeneration and regrowth of SON and PVN axons following hypophysectomy in the endocrine hypothalamus of the rat. Our data support the hypothesis that NO may serve as a second messenger molecule that may act in some fashion to govern not only the process of central regeneration and regrowth of magnocellular (SON/PVN) axons into the median eminence, neural stem, and neural lobe (the neurohypophyseal system) but may also influence the regeneration of neurites into new neuroanatomical domains such as the adjacent lumen of the third cerebral ventricle. We have demonstrated a distinct temporal relationship between injury (axotomy) of SON/PVN axons and the establishment of new neurovascular zones following hypophysectomy with the up-regulation of NOS. This up-regulation appears to correlate well with successful regeneration in the mammalian neurohypophyseal system. We have also successfully inhibited axonal regeneration with the use of nitroarginine, a competitive antagonist of NO. NOS up-regulation attendant to regeneration of SON and PVN axons may have inestimable clinical implications, particularly with respect to closed head injury and cerebral contusion that involves the mechanical shearing of the infundibular stalk. In addition, this investigation has reaffirmed that large numbers of bona fide neurons migrate and emerge upon the floor of the adjacent third cerebral ventricle shortly following hypophysectomy (within 2 weeks). The origin and mechanisms of neuronal migration and plasticity following hypophysectomy are the subject of interpretation and discussion in this investigation.