The myogenic response of cerebral arteries is responsible for intravascular pressure-dependent control of blood flow to the brain1. This mechanism depends on cellular processes intrinsic to vascular smooth muscle cells (VSMCs) including: 1) Ca2+ influx, Ca2+- dependent activation of myosin light chain kinase, and phosphorylation of the 20 kDa regulatory light chains (LC20) of myosin, and 2) Rho-associated kinase (ROK)-dependent phosphorylation of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase and suppression of the phosphatase activity2. Inappropriate regulation of these intrinsic mechanisms leading to dysfunctional control of cerebral arterial diameter may contribute to ischemic and hemorrhagic stroke in type 2 diabetes (T2D)3. Here, we used the Goto-Kakizaki (GK) rat model of T2D to determine if specific alterations in LC20 and MYPT1 phosphorylation contribute to dysfunctional control of cerebral arterial diameter. Rats were killed by halothane inhalation and exsanguination. Endothelium-denuded middle and posterior cerebral arteries of 8-10 and 18-20 week old GK and Wistar (WR) control rats were studied by pressure myography to detect pressure-dependent alterations in diameter (n=8), and by western blotting to quantify changes in LC20 and MYPT1 phosphorylation levels at 10, 60 and 120 mmHg (n=6)4. Values were expressed as mean±SEM and compared by ANOVA. Pressure-dependent increases in phospho-LC20 (from 26±0.5% to 38±1% and 49±1%; p<0.05) and phospho-MYPT1-T855 (from 1.0 to 2.0±0.15 and 2.8±0.18 normalized units; p<0.05) were detected at 10, 60 and 120 mmHg in vessels of 8-10 and 18-20 wks WR. Vessels of 8-10 wks GK had a compromised myogenic response, with enhanced constriction at low pressure and forced dilation at >100 mmHg, that progressed to a loss of myogenic tone in 18-20 wks GK. Phospho-MYPT1-T855 was elevated at 10 mmHg in 8-10 wks GK compared to WR (1.42±0.04-fold; p<0.05), and no increase was detected with pressure elevation to 60 or 120 mmHg (1.54±0.1 and 1.45±0.12). Phospho-LC20 was similarly elevated at 10 mmHg (35±1.8%) in 8-10 wks GK vessels, but no increase was detected on pressure elevation to 60 or 120 mmHg (40±1.9% and 37±2%, respectively). Phospho-LC20 and phospho-MYPT1-T855 levels of 18-20 wks GK and WR were not different at 10 mmHg, but a pressure-dependent increase in phosphorylation was not detected in GK. Inhibition of ROK with H1152 (0.5 µM) abolished the enhanced constriction at low pressure in 8-10 wks GK and suppressed the residual tone of vessels from 18-20 wks GK. Our findings suggest that a progressive dysfunctional control of ROK activity, and consequently inappropriate MYPT1 and LC20 phosphorylation, contribute to the abnormal myogenic response in cerebral resistance arteries of T2D GK rats.