The cytoskeleton of cerebral microvascular endothelial cells is a critical determinant of blood-brain barrier (BBB) function. Barrier integrity appears to be particularly sensitive to the phosphorylation state of specific residues within myosin regulatory light chain (RLC), one of two accessory light chains of the myosin II motor complex. Phosphorylation of myosin RLC by myosin light chain kinase (MLCK) has been implicated in BBB dysfunction associated with alcohol abuse and hypoxia, whereas dephosphorylation may enhance BBB integrity following exposure to lipid-lowering statin drugs. Using immunohistochemistry we provide evidence of widespread myosin II RLC distribution throughout the cerebral vasculature of the mouse. Light microscopy revealed immunolocalization of myosin II RLC protein in the endothelium of brain capillaries, the endothelial cell layer of arterioles and in association with venules. Immunolabeling of myosin RLC in non-muscle endothelial cells could be distinguished from myosin RLC immunoreactivity associated with the smooth muscle layer of the tunica media in larger muscular arterioles. These findings support an emerging role for myosin II RLC as a component of the actomyosin cytoskeleton of cerebral endothelial cells with the potential to contribute to the selective vulnerability of the brain in vivo.