Further aminopeptidase activity on Ang produces Ang and Ang, which may also have biological activity. A new dimension was added to the brain RAS with the discovery of a novel non-AT1, non-AT2 binding site for Ang II. Initial studies of this novel binding site could not ascertain its function and it was hypothesized to be either a signaling or clearance receptor, or a peptidase. Ang II binding site was the use of a mouse strain in which the neurolysin gene was knocked out. Expression of the non-AT1, non-AT2 binding site was dramatically decreased in the brains of the neurolysin knockout mouse strain compared to wild-type mice. Distribution was then examined using quantitative densitometric autoradiography. A qualitative sampling of this autoradiographic analysis was included in our previous publication. Additionally, we examined the distribution and concentration of AT1 and AT2 receptors in the brains of the neurolysin knockout mouse strain in comparison to wild-type mice using the same methodology. Neurolysin binding was widespread throughout the brain, showing only a 3.5-fold difference in density among sampled brain regions, in contrast to the discrete localization of AT1 and AT2 receptors in the mouse brain. Indeed, neurolysin has a broad array of substrates, thus its distribution beyond that of the angiotensin receptors is not unexpected. With respect to the functional significance of neurolysin to the brain RAS, the INCB28060 significant reduction in AT2 receptor binding suggests that neurolysin plays a role in maintaining AT2 receptor expression in the brain. There are two comprehensive studies of the regional density of mouse brain AT1 and AT2 receptors. While they show agreement with the regions that contain AT1 receptor binding, the relative densities in 7 overlapping regions were not significantly correlated. The distribution of AT2 receptor binding in this study varied from the comprehensive studies with respect to AT2 receptor binding in the hypothalamus. However a limited study of the hypothalamic AT2 receptor binding as well as an immunohistochemical analysis indicated the presence of AT2 receptors in the paraventricular nucleus of the hypothalamus in agreement with this study. The upregulation of brain AT2 receptors is yet another indicator of a potential beneficial effect of neurolysin, since increased expression and/or stimulation of brain AT2 receptors is associated with neuronal protection. The lateral ventricular enlargement observed in the neurolysin knockout brains and the presence of neurolysin in the choroid plexus may indicate a role for neurolysin in the blood brain barrier and blood-cerebrospinal fluid permeability. This effect was limited to the lateral ventricles as no changes in the third and fourth ventricles or the cerebral aqueduct were observed between strains. This indicates that reduced flow of CSF through the cerebral aqueduct is not a cause of the lateral ventricle enlargement. Peptidases in the cerebral microvasculature and choroid plexus decrease the effects of circulating peptides on the cerebral microvasculature and help prevent blood-borne peptides from entering the brain via metabolic inactivation.