Antibodies to chicken fast skeletal muscle (pectoralis) a-actinin and to smooth muscle (gizzard) a-actinin were absorbed with opposite antigens by affinity chromatography, and four antibody fractions were thus obtained: common antibodies reactive with both pectoralis and gizzard a-actinins ([C]anti-P a-An and [C]anti-G a-An), antibody specifically reactive with pectoralis a-actinin ([S]anti-P a-An), and antibody specifically reactive with gizzard a-actinin ([S]anti-G a-An). In indirect immunofluorescence microscopy, (C)anti-P aAn, (S)anti-P a-An, and (C)anti-G a-An stained Z bands of skeletal muscle myofibrils, whereas (S)anti-G a-An did not. Although (S)anti-G a-An and two common antibodies stained smooth muscle cells, (S)anti-P u-An did not. We used (S)anti-P a-An and (S)anti-G a-An for immunofluorescence microscopy to investigate the expression and distribution of skeletal-and smoothmuscle-type a-actinins during myogenesis of cultured skeletal muscle cells. Skeletal-muscletype a-actinin was found to be absent from myogenic cells before fusion but present in them after fusion, restricted to Z bodies or Z bands. Smooth-muscle-type ~-actinin was present diffusely in the cytoplasm and on membrane-associated structures of mononucleated and fused myoblasts, and then confined to membrane-associated structures of myotubes. Immunoblotting and peptide mapping by limited proteolysis support the above results that skeletalmuscle-type a-actinin appears at the onset of fusion and that smooth-muscle-type a-actinin persists throughout the myogenesis. These results indicate (a) that the timing of expression of skeletal-muscle-type a-actinin is under regulation coordinate with other major skeletal muscle proteins; (b) that, with respect to expression and distribution, skeletal-muscle-type a-actinin is closely related to a-actin, whereas smooth-muscle-type a-actinin is to 3'-and ~-actins; and (c) that skeletal-and smooth-muscle4ype a-actinins have complementary distribution and do not co-exist in situ.a-Actinin, which was discovered by Ebashi and Ebashi (12), is a structural protein of Z bands in skeletal muscle myofibrils (33). It is also located near the fascia adherens of intercalated disks as well as in Z bands in cardiac muscle (44). In smooth muscle, it is present in cytoplasmic dense bodies and membrane-associated dense plaques (21,41). Proteins immunologically and biochemically related to a-actinin have been isolated from several nonmuscle tissues or cells: bovine brain (42), Ehrlich tumor cells (36,37), plasma membranes of sarcoma 180 ascites cells (46), HeLa cells (6), human platelets (40), rat liver (29), and porcine kidney (28). Many of these proteins, if not all, are Ca 2+ sensitive, i.e., are inhibited from cross-linking actin filaments by Ca 2+, different from any of the muscle a-actinins. Immunofluorescence microscopy, immunoelectron microscopy, and microinjection using fluorescence-labeled a-actinin have revealed that a-actinin or proteins immunologically related to a-actinin are also present in cultur...