Multifunctional type II Ca2+/calmodulin-dependent protein kinase purified from rat brain cytosol was found to bind to actin filaments in vitro. The binding was saturable, and the dissociation constant for the binding was determined to be about 4 X 10(-8)M. Electron microscopic observation indicated that the kinase binds to the side of actin filaments. Calmodulin inhibited the binding of the kinase to actin filaments in a Ca2+-dependent manner. The Ca2+/calmodulin-regulated binding of the kinase to actin filaments revealed here may be important for the substrate recognition of the kinase.