Animal models are of pivotal importance in vascular physiology, allowing the application of different ‘challengers’ for the in vivo study of regulatory phenomena, for example drugs to modulate vascular function. The effect of these challengers is best observed with noninvasive perfusion assessment techniques. Our objective was to characterize the microcirculatory reactivity to phentolamine (PL). A group of six C57/BL6 male mice (12 w.o.) under a ketamine (K, 137.5 mg/kg) and xylazine (X, 11 mg/kg) mixture received two IP doses of PL (2.5 mg/kg). Animal experiments were performed in accordance with the EU guidelines on animal welfare and complied with principles and standards for reporting animal experiments. Perfusion was assessed with laser Doppler flowmetry (LDF), collected on a random hindlimb and decomposed with the wavelet transform (WT) for the inspection of the respective spectral components – cardiac, respiratory, myogenic, sympathetic, endothelial NO-dependent (NOd) and endothelial NO-independent (NOi). Both PL doses increased perfusion, although without statistical significance. The first PL dose caused the significant decrease of the myogenic activity, together with the significant increase of the sympathetic and NOd activities. The second PL dose increased the myogenic activity and decreased the NOd, although baseline values were not reached. PL acts as an alpha-1 receptor antagonist, which is in line with the observation of perfusion increase. However, this mechanism contrasts with the observed increase in sympathetic activity. Therefore, these results suggest that, under ketamine-xylazine anesthesia, PL administration evokes mainly an indirect vascular effect, resulting from the combined effect of both the myogenic and endothelial activities.