Myosin, the predominant protein within the myofibrils of skeletal and cardiac muscle occurs in several isomorphic (isoenzymic) forms. Evidence is accumulating that early in development two or more of these isomorphs can coexist within the same cells, but with progressive development the individual myosin types become segregated into their characteristic adult locations. Detection of myosin isoenzymes is often done immunologically with appropriately cross-absorbed xenogenic antisera. Problems have often arisen with the specificity of these antisera and uncertainties remain about the number of different structural genes for myosin heavy and light chains which are truly expressed in different muscle cells. Also, it is unclear if different myosin isomorphs coassemble within common myofibrils, sarcomeres and thick filaments. Using the newly developed technique of Kohler and Milstein (1975), Nature 256:495, we have generated 18 hybridoma lines which secrete monoclonal antibodies to myosin isolated from adult chick breast muscle. Three of these antibody preparations for breast myosin where as the other 15 crossreact to varying degrees with other myosin isoenzymes. In this application, we propose to generate additional monoclonal antibodies specific to the adult and embryonic myosins from cardiac, ALD and pectoralis major muscles of the chicken. Specificities and binding affinities will be compared by radioimmune assay and immunofluorescence. Location of antigenic determinants within myosin molecules will be established by antibody binding to denatured subunits, proteolytic fragments of heavy chains and electron microscopic immunocytochemistry. Detection of isomorphic variants with embryonic heart and skeletal muscle will then be performed by double label immunofluorescence with cell cultures and frozen sections of embryonic material.