OBJECTIVE Pial arterioles transiently dilate during cortical spreading depression (CSD), although the mechanisms are unclear. We tested the hypothesis that increased production of nitric oxide (NO) promotes arteriolar dilation. METHODS Urethane-anesthetized rabbits were equipped with cranial windows, and the diameter (reported in micrometers) of a pial arteriole was determined via intravital microscopy. In each rabbit, a baseline CSD was elicited by microapplication of KCl onto the cortex, and resultant pial arteriolar dilation was measured. Either 100 mumol/L N omega-nitro-L-arginine methyl ester (L-NAME) or 50 mumol/L NG-nitro-L-arginine (L-NA), both competitive NO synthase inhibitors, was then applied to the brain surface. A CSD was elicited as before. The L-NAME and L-NA were then removed by artificial cerebrospinal fluid washes. An additional CSD was induced with KCl as before. RESULTS Control CSD in the L-NAME group dilated pial arterioles; baseline diameter, 66 +/- 7 mm, with CSD = 106 +/- 8 mm (59% increase). After topically applied L-NAME, CSD dilated pial arterioles less: baseline diameter, 61 +/- 7 mm, with CSD = 77 +/- 6 mm (26% increase), P < .05 compared with control CSD diameter. Topical L-NA had similar effects on CSD: control CSD dilated pial arterioles 51%; after topical L-NA, only 14% (P < .05). After removal of L-NAME or L-NA, CSD-induced pial arteriolar dilation was similar to original control values. CONCLUSIONS The reversible inhibition of CSD-induced pial arteriolar dilation by either L-NAME or L-NA suggests that NO contributes to arteriolar dilation observed with CSD.