Pial arterioles do not express N-methyl-D-aspartate (NMDA) receptors but dilate in response to topical NMDA application. We explored the mechanism underlying NMDA-mediated responses in murine pial arterioles (11-31 µm) using a closed cranial window preparation, and found that arteriolar dilation was not concentration-dependent. Pial arteriolar diameter abruptly increased within 3 minutes of superfusing 50 or 100 µM NMDA. Dilation reached a peak within 1 minute (46±14%) and then declined to a plateau (28±13%) for the duration of superfusion. While a higher concentration (200 µM) did not produce further dilation, lower concentrations (1-10 µM) did not dilate the arterioles at all. MK-801 (10 µM) abrogated the dilation response, whereas L-NA (1 mM) attenuated the peak and abolished the sustained dilation during NMDA superfusion. We determined that NMDA-induced pial arteriolar responses were evoked by cortical spreading depression, because, abrupt vasodilation during 50 or 100 µM NMDA superfusion was associated with a large negative DC shift and ECoG suppression that spread from the superfusion window to distant cortical areas. Our data suggest that the responses of pial arterioles to NMDA are caused in part by neurovascular coupling due to cortical spreading depression.