The purpose of the present study was to compare the effects of endothelium-dependent acetylcholine (ACh) and endothelium-independent sodium nitroprusside (SNP) vasodilators on the oscillatory components of the cutaneous blood perfusion signals in humans. The unstimulated basal blood perfusion and the blood perfusion during iontophoretically delivered ACh and SNP were measured using laser Doppler flowmetry (LDF). The wavelet transform was calculated before spectral analysis of the measured signals. In the frequency interval from 0.0095 to 1.6 Hz the LDF signal consists of oscillations with five different characteristic frequencies. In addition to the cardiac (1 Hz) and respiratory (0.3 Hz) rhythms, three other oscillations in the regions around 0.1, 0.04, and 0.01 Hz were detected. The oscillations with the different frequencies were observed in unstimulated blood flow and also during stimulation with ACh and SNP. Compared to the unstimulated blood flow, both ACh and SNP increased the mean amplitude of the total spectrum (P< 0.005 for both substances). The only significant difference between the effects of ACh and SNP was observed in the amplitude of oscillations with the frequency of around 0.01 Hz. ACh increased the absolute amplitude of this frequency to a greater extent than SNP in athletes (P= 0.03), whereas only a trend was observed in controls (P= 0.2). The relative amplitude, defined as the ratio between the absolute amplitude of a particular frequency interval and the mean amplitude of the total spectrum, was also higher for ACh compared to SNP both in controls (P= 0.008) and in athletes (P= 0.004), only for oscillations with the frequency of around 0.01 Hz. We conclude that ACh selectively influences the oscillatory component of around 0.01 Hz in the cutaneous blood perfusion signal to a greater extent than SNP. This finding indicates that endothelium-mediated vasodilatation is manifested as oscillations with a repetition time of approximately 1 min. The mechanisms for the endothelial dependency of this frequency remain to be elucidated. Our data indicate that spectral analysis based on wavelet transform of the cutaneous perfusion signal can be used clinically to investigate endothelial function. The described noninvasive method might be used to evaluate endothelial function for research, for diagnostic purposes, and maybe also to assess effects of therapy in cardiovascular diseases.