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Botulinum toxin-A (BTX) has numerous cosmetic and therapeutic applications. Our previous studies have found that BTX augments pedicled flap survival through both vasodilatory effects and attenuation of the inflammatory response to ischemia in the rat. This study examines the effect of chronic BTX on microcirculatory vascular tone and its response to acute topical vasodilators in muscle flaps.The spinotrapezius muscle of Sprague-Dawley rats underwent a single 2-week pretreatment of 0.2 mL saline either with (n = 5) or without (n = 5) 2u BTX. After surgical elevation, an arcade arteriole was observed using a video caliper device. Vessel diameter was measured at 30-second intervals after sequential superfusion of nitroglycerin (100 and 200 μg/mL), multiple concentrations of lidocaine, and a combination of adenosine (10 μM) and nitroprusside (10 μM) to induce maximum dilation.Baseline and dilation diameters were expressed as ratios of pharmacologically induced maximum dilation, whereas percent dilation was defined as the change in diameter over baseline diameter. We found a significant increase in resting diameter with BTX pretreatment (P = 0.0028). Compared with the control group, mean baseline diameter was 15% greater, and percent dilation was 25% less in BTX-pretreated flaps. There was no significant relationship between BTX pretreatment and dilation diameter (P = 0.2895) after adjusting for the effect of acute vasodilators.Pretreatment with BTX may induce the arteriolar resting diameter to be closer to their maximum potential diameter. Additionally, BTX does not display a synergistic effect with topical vasodilators on vasodilation.