Cerebral microvascular endothelial cells (CMVECs) have recently been implicated as targets of excitotoxic injury by L-glutamate (L-glut) or N-methyl-D-aspartate (NMDA) in vitro. However, high levels of L-glut do not compromise the function of the blood brain barrier in vivo. We sought to determine whether primary cultures of rat and piglet CMVECs or cerebral microvascular pericytes (CMVPCs) are indeed sensitive to L-glut or NMDA. Viability was unaffected by 8h exposure to 1-10 mM L-glut or NMDA in CMVECs or CMVPCs isolated from both species. Furthermore, neither 1mM L-glut nor NMDA augmented cell death induced by 12h oxygen-glucose deprivation in rat CMVECs or by 8h medium withdrawal in CMVPCs. Additionally, transendothelial electrical resistance of rat CMVEC-astrocyte co-cultures or piglet CMVEC cultures were not compromised by up to 24h exposure to 1mM L-glut or NMDA. The Ca²⁺-ionophore calcimycin (5µM), but not L-glut (1mM) increased intracellular Ca²⁺ levels in rat CMVECs and CMVPCs assessed with Fluo4 AM-fluorescence using confocal microscopy. CMVEC-dependent pial arteriolar vasodilation to hypercapnia and bradykinin was unaffected by intracarotid infusion of L-glut in anesthetized piglets using closed cranial window/intravital microscopy. We conclude that cerebral microvascular cells are insensitive and resistant to glutamatergic stimuli in accordance with their in vivo role as regulators of potentially neurotoxic amino acids across the blood brain barrier.