This study evaluates the validity of the transfer function analysis of spontaneous fluctuations of arterial blood pressure (ABP) and blood flow velocity of the middle cerebral artery (MCAFV) as a simple, convenient method to assess human cerebral autoregulation in patients with carotid stenosis. Eighty-three consecutive patients with various degrees of carotid stenosis and 37 healthy controls were enrolled. The carotid stenosis was graded based on the diagnostic criteria of duplex ultrasound. Instantaneous bilateral MCAFV and ABP of all participants were assessed noninvasively using transcranial Doppler sonography and the servocontrolled infrared finger plethysmography, respectively. Spectral analyses of ABP and MCAFV were performed by fast Fourier transform. The fluctuations in ABP as well as in MCAFV were diffracted into three components at specific frequency ranges designated as high-frequency (HF; 0.15 to 0.4 Hz), low-frequency (LF; 0.04 to 0.15 Hz), and very low-frequency (VLF; 0.016 to 0.04 Hz). Cross-spectral analysis was applied to quantify the coherence, transfer phase, and magnitude in individual HF, LF, and VLF components. Transcranial Doppler CO2 vasomotor reactivity was measured with 5% CO2 inhalation. The LF phase angle (r=-0.53, P<0.001); magnitude of VLF (r=-0.29, P=0.002), LF (r=-0.35, P<0.001), and HF (r=-0.47, P<0.001); and CO2 vasomotor reactivity (r=-0.66, P<0.001) were negatively correlated with the severity of stenosis. Patients with unilateral high-grade (greater than 90% stenosis) carotid stenosis demonstrated significant reduction in LF phase angle (P<0.001) and HF magnitude (P=0.018) on the ipsilateral side of the affected vessel compared with their contralateral side. The study also revealed a high sensitivity, specificity, and accuracy using LF phase angle and HF magnitude to detect a high-grade carotid stenosis. A strong correlation existed between the LF phase angle and the CO2 vasomotor reactivity test (r=0.62, P<0.001), and the correlation between the HF magnitude and the CO2 vasomotor reactivity (r=0.44, P<0.001) was statistically significant as well. We conclude that transfer function analysis of spontaneous fluctuations of MCAFV and ABP could be used to identify hemodynamically significant high-grade carotid stenosis with impaired cerebral autoregulation or vasomotor reserve.