This study aimed to analyze the frequency components present in spontaneous rhythmic diameter changes in rat pial arterioles. Pial microcirculation was visualized by fluorescence microscopy. Rhythmic luminal variations were evaluated via computer-assisted methods. Spectral analysis was carried out on 30-min recordings under baseline conditions and after administration of acetylcholine (Ach), papaverine (Pap), Nω-nitro-L-arginine (L-NNA) prior to Ach, indomethacin (INDO), INDO prior to Ach, charybdotoxin and apamin, and charybdotoxin and apamin prior to Ach. Under baseline conditions all arteriolar orders showed 3 frequency components in the ranges of 0.0095-0.02, 0.02-0.06, and 0.06-0.2 Hz, another 2 in the ranges of 0.2-2.0 and 2.5-4.5 Hz, and another ultra-low-frequency component in the range of 0.001-0.0095 Hz. Ach caused a significant increase in the spectral density of the frequency components in the range of 0.001-0.2 Hz. Pap was able to slightly increase spectral density in the ranges of 0.001-0.0095 and 0.0095-0.02 Hz. L-NNA mainly attenuated arteriolar responses to Ach. INDO prior to Ach did not affect the endothelial response to Ach. Charybdotoxin and apamin, suggested as endothelium-derived hyperpolarizing factor inhibitors, reduced spectral density in the range of 0.001-0.0095 Hz before and after Ach administration. In conclusion, regulation of the blood flow distribution is due to several mechanisms, one of which is affected by charibdotoxin and apamin, modulating the vascular tone.