The binding sites of five monoclonal antibodies against myosin of Dictyostelium discoideum have been mapped. These antibodies bind to the tail region of the myosin molecule. By rotary shadowing, images of myosin-antibody complexes were obtained in which the mean distance of the midpoint of an antibody molecule from the myosin heads was localized with a precision better than 2 nm (90% confidence limit). Other quantitative data extracted from electron micrographs provided information on the stoichiometry of antibody-myosin interaction. Certain antibodies interacted with myosin molecules only at a ratio of 1:1. Other antibodies formed complexes of two molecules bound to homologous sites on a double-stranded myosin tail. Affinities were estimated and the abilities of different antibodies to cross-connect two myosin molecules were evaluated.