The primary purposes of this study were to: (1) examine selected properties of the intermediate filament-associated protein (IFAP), synemin, (2) identify synemin (or a synemin homolog) in adult mammalian muscle, and (3) examine synemin's ability to interact with proteins located at the myofibrillar Z-line. An integrated biochemical, immunological, and ultrastructural approach was used;Synemin (230 kD) was solubilized from an actomyosin-extracted avian smooth muscle residue and purified by chromatography on two hydroxyapatite columns and one DEAE-Sephacel column in the presence of 6 M urea. Renatured, soluble synemin was obtained by removal of the urea by dialysis against 10 mM Tris-HCl, pH 8.5. Electron microscopy of negatively stained specimens revealed that synemin in 10 mM Tris-HCl, pH 8.5, is spherical in nature (diameter ~11 nm), and chemical crosslinking experiments showed that synemin molecules exist primarily as dimers. Synemin and desmin have similar pH- and ionic strength-dependent solubility properties, but desmin forms intermediate filaments (IFs) and synemin forms complex aggregates under physiological-like conditions. Synemin's wide distribution in adult avian and mammalian skeletal, cardiac, and smooth muscles was shown by Western blot analysis and by immunofluorescence labeling of isolated myofibrils and of muscle cryosections. Double-labeling experiments with conventional immunofluorescence, confocal scanning laser microscopy, and computer-assisted image analysis showed that desmin and synemin colocalize at the myofibrillar Z-lines in a punctate pattern;Synemin's ability to interact with desmin was examined by negative staining and immunogold electron microscopy as well as by immunoblot overlay assays. Purified desmin self-assembles into long (>1 [mu]m) IFs when dialyzed against physiological-like buffers; however, desmin's ability to assemble into these long IFs decreases as the relative amount of synemin to desmin increases. The smallest, full width (~10 nm) IF assembly intermediate formed in the presence of synemin was ~50-70 nm long. Immunogold labeling experiments indicated that synemin binds along the desmin IFs, or at points of filament intersection. Solid-phase binding assays indicated that synemin can bind to desmin and to [alpha]-actinin;These results, taken in toto, indicate that synemin (or a homolog) exists in mammalian muscles, that synemin copolymerizes with, or binds along the length of, desmin IFs, and suggest that synemin may serve as a cytoskeletal crosslinking component between IFs and myofibrils in muscle cells.