The purpose of this study was to determine whether spontaneous oscillations in blood flow (relative red blood cell flux) measured by laser Doppler flowmetry (LDF) in the cat optic nerve head were related to fluctuations in nitric oxide (NO) measured with electrochemical sensors (n = 16 cats). Power spectral densities for the magnitude and frequency of LDF and NO fluctuations were determined by discrete Fourier transform analysis. Complex behavior was found for both LDF and NO oscillations with broad spectra containing peaks at multiple frequencies. Most of the power was in the low-frequency range (<10 cycles/min). Spectra were also obtained after administering NO synthase inhibitors (l-nitroarginine, L-NA, n = 6 cats; l-nitroarginine methyl ester, L-NAME, n = 5 cats). Both inhibitors caused a decrease in blood flow, basal NO levels, and amplitude of NO fluctuations. There was little change in amplitude for blood flow oscillations, with some enhancement at the lowest frequencies. We conclude that NO is not required for vasomotion and that spontaneous, low-frequency NO fluctuations observed in the cat optic nerve head are a passive phenomenon caused by natural variations in shear stresses.