The role of nitric oxide (NO) in the regulation of optic nerve head (ONH) blood flow was investigated from direct electrochemical measurements of NO and simultaneous measurements of ONH blood flow by laser Doppler flowmetry during increased neuronal activity with diffuse luminance flickering light stimuli. For normal physiological conditions, there was a consistent increase in ONH blood flow with each flicker stimulus (n = 10 cats). Simultaneous NO measurements were made in the vitreous humor immediately in front of the ONH (n = 6 cats). During flicker, NO increased by 88 +/- 23 nM (P < 0.05) above basal NO levels. Measurements were repeated after inhibiting NO synthase by intravenous infusion of either NG-nitro-L-arginine (n = 5 cats) or NG-nitro-L-arginine methyl ester (n = 5 cats). After inhibition, NO levels were significantly reduced and both ONH blood flow and NO responses to flicker were significantly attenuated. We conclude that NO plays a key vasodilatory role in the ONH, modulating blood flow during greater neuronal activity with flicker stimuli.