The intestinal epithelial cell brush border (BB) is a useful model for nonmuscle cell motility. We studied regulation of BB motility by analyzing myosin phosphorylation and its association with the cytoskeleton. Our results demonstrate that myosin associates with the cytoskeleton only when it is dephosphorylated. Myosin light chain kinase substrates release myosin, phosphorylated and in the form of filaments, from the cytoskeleton. Although ITP and GTP serve as myosin ATPase substrates, they do not cause BB contraction, myosin release, or phosphorylation. Brush border contraction occurs with ATP or with a mixture of ITP and ATP gamma S. Therefore, phosphorylation regulates myosin association with the cytoskeleton, myosin is not bound at the actin-myosin binding site, and when phosphorylated, myosin forms filaments for movement.