An aging rat model of chronic brain hypoperfusion (CBH) that mimics human mild cognitive impairment (MCI) was used to examine the role of nitric oxide synthase (NOS) isoforms on spatial memory function. Rats with CBH underwent bilateral common carotid artery occlusion (2-vessel occlusion (2-VO)) for either 26 or 8 weeks and were compared with nonoccluded sham controls (S-VO). The neuronal and endothelial (nNOS/eNOS) constitutive inhibitor nitro-L-arginine methyl ester (L-NAME) 20 mg/kg was administered after 26 weeks for 3 days to 2-VO and S-VO groups and spatial memory was assessed with a modified Morris watermaze test. Only 2-VO rats worsened their spatial memory ability after L-NAME. Electron microscopic immunocytochemical examination using an antibody against eNOS showed 2-VO rats had significant loss or absence of eNOS-containing positive gold particles in hippocampal endothelium and these changes were associated with endothelial cell compression, mitochondrial damage and heavy amyloid deposition in hippocampal capillaries and perivascular region. In the 8-week study, three groups of 2-VO rats were administered an acute dose of 7-NI, aminoguanidine or L-NIO, the relatively selective inhibitors of nNOS, inducible NOS and eNOS. Only rats administered the eNOS inhibitor L-NIO worsened markedly their watermaze performance (P = 0.009) when compared with S-VO nonoccluded controls. We conclude from these findings that vascular nitric oxide derived from eNOS may play a critical role in spatial memory function during CBH possibly by keeping cerebral perfusion optimal through its regulation of microvessel tone and cerebral blood flow and that disruption of this mechanism can result in spatial memory impairment. These findings may identify therapeutic targets for preventing MCI and treating Alzheimer's disease.