Synemin is a cytoskeletal protein originally identified as an intermediate filament (IF)-associated protein because of its colocalization and copurification with the IF proteins desmin and vimentin in muscle cells. Our sequencing studies have shown that synemin is an unusually large member (1,604 residues, 182,187 Da) of the IF protein superfamily, with the majority of the molecule consisting of a long C-terminal tail domain. Molecular interaction studies demonstrate that purified synemin interacts with desmin, the major IF protein in mature muscle cells, and with alpha-actinin, an integral myofibrillar Z-line protein. Furthermore, expressed synemin rod and tail domains interact, respectively, with desmin and alpha-actinin. Analysis of endogenous protein expression in SW13 clonal lines reveals that synemin is coexpressed and colocalized with vimentin IFs in SW13.C1 vim+ cells but is absent in SW13.C2 vim- cells. Transfection studies indicate that synemin requires the presence of another IF protein, such as vimentin, in order to assemble into IFs. Taken in toto, our results suggest synemin functions as a component of heteropolymeric IFs and plays an important cytoskeletal cross-linking role by linking these IFs to other components of the cytoskeleton. Synemin in striated muscle cells may enable these heterofilaments to help link Z-lines of adjacent myofibrils and, thereby, play an important role in cytoskeletal integrity.