Pericytes are contractile cells located on microvessels, and are found at highest density in the central nervous system. In the brain, pericytes dilate capillaries in response to neuronal activity, targeting blood flow to active neurons (Hall et al., 2014). However, following ischaemia, they irreversibly constrict capillaries and die in rigor. Pericytes also play many other critical roles in CNS development, physiology, and pathology, yet discord regarding their identification persists. We investigated the anatomy, physiology, and pathological dysfunction of pericytes. Methods: We used NG2-DsRed mice (n=13) and Sprague-Dawley rats for experiments. Data are presented as mean ± SEM. Acute brain slices were prepared from P12 rats (n=25) for physiological experiments and P21 (n=35) rats for ischaemia experiments. When examining the pericyte response to ischaemia in vivo, we used the intraluminal filament model of middle cerebral artery occlusion (MCAO) to simulate stroke. Rats (male, 250-300g, n=3) were anaesthetised with isoflurane (4% for induction) prior to maintenance with urethane (1.5g/kg IP) for surgery and MCAO. Ischaemia was induced for 90mins. Two hours after MCAO onset animals were sacrificed. Intravascular fluorescent gel perfusion was used to assess brain capillary diameter near pericytes in fixed tissue. MCAO animals were compared with naïve controls (n=3). Results: Pericytes are a distinct cell population and were readily distinguished from endothelial cells and juxtavascular microglia by their expression of the proteoglycan NG2, the receptor tyrosine kinase PDGFRβ, and their being surrounded by the basement membrane marker isolectin B4. Pericytes extend contractile processes along and around the endothelial cells forming capillaries. We found 33±3% of brain pericytes were contacted by microglial processes, potentially mediating surveillance of the blood-brain barrier. In cerebellar slices, NO (delivered as DETA-NONOate, 100μM) and PGE2 (1μM) dilated molecular layer capillaries by 13.5±2.3% and 17.7±3.8% of baseline diameter, respectively. Electrical stimulation of the cerebellar parallel fibres dilated capillaries by 14.6±3.4%. Dilation was mediated by PGE2 acting at EP4 receptors: dilation was blocked by the specific EP4 receptor blocker L161,982 (1μM, p=0.01, t-test). Capillary constriction at pericytes in brain slices occurred in ~15mins when ATP synthesis was inhibited with iodoacetate (2mM) and antimycin (25μM). After MCAO, capillaries were more constricted near pericytes compared to control (p=0.035, t-test). In ischaemic brain slices, capillary constriction was significantly delayed by blocking voltage-dependent calcium channels (nimodipine, 100μM, p=0.006, t-test) and 20-HETE synthesis (HET0016, 1μM, p=0.004, t-test), suggesting the use of such agents in ischaemic stroke should be re-evaluated.