Intro: Pressure-dependent myogenic responses play a significant role in modulating autoregulation of resistance arteries, especially in cerebral circulation [1]. Increasing evidence suggests a potential physiological role for NADPH-oxidase and reactive oxygen species (ROS) in pressure-dependent myogenic tone [2]. There may also be potential link between ROS and actin cytoskeletal dynamics [3]. In this study, we investigated the potential role of NADPH-oxidase/ROS and actin polymerization in acute pressure-dependent vascular contraction. Method: Adult male Sprague-Dawley rats (12 weeks, 250~300g) were asphyxiated using CO2 and euthanized by cervical dislocation. Pressure-dependent responses were recorded in isolated mid cerebral arteries (MCA; Diameter 150 ± 7.81 μm) using pressure myography. Pressure-dependent responses were studied by pressure-step (40 to 80 to 120 mmHg). Confocal microscopy was used to visualize staining of F-actin using phalloidin-FITC (1μM) in pressure-fixed vessel. Statistics analysis was measured by two-way ANOVA for repeated measure and values presented as mean ± SEM. Results & Discussion: Pressure-dependent myogenic tone in MCA control was 11.46 ± 3.05, 18.72 ± 2.88 and 23.75 ± 2.61 % at 40, 80 and 120mmHg, respectively (n=14). Pressure-dependent myogenic tone was significantly reduced following NAC, DPI and Cyto D incubation. The antioxidant N-acetylcysteine (NAC, 10mM, n=4) reduced myogenic tone to 7.72 ± 1.31, 7.44 ± 2.81, 8.06 ± 1.44 % at 40, 80 and 120mmHg respectively. The NADPH-oxidase inhibitor diphenyleneiodonium (DPI, 10μM, n=3) reduced myogenic tone to 5.93 ± 1.53, 2.00 ± 2.33, -1.15 ± 2.25 % at 40, 80 and 120mmHg, respectively. Likewise, the actin polymerization inhibitor Cytochalasin D (Cyto D, 5μM, n=4) reduced myogenic tone to 9.52 ± 4.32, 3.79 ± 0.67, 0.45 ± 1.78 % at 40, 80 and 120mmHg, respectively. Time-control experiments showed sustained pressure-dependent myogenic tone. NAC and DPI did not have any effect on high-K+ PSS (60mM) constriction. The confocal study showed that there was a marked increase of actin polymerization in vascular smooth muscle (VSM) at 120mmHg compared to 40mmHg. The effect of Cyto D greatly reduced the fluorescence measured at 120 mmHg. This further confirmed a dynamic actin polymerization contribution to the pressure-dependent myogenic tone mechanism in the cerebral arteries. Conclusion: These data suggest that the activation of NADPH oxidase/ROS/ actin polymerization as part of the acute pressure-dependent myogenic constriction mechanism.