In skeletal muscles, the sarcolemma is possibly stabilized and protected against contraction-imposed stress by intermediate filaments (IFs) tethered to costameric sarcolemma. Although there is emerging evidence that plectin links IFs to costameres through dystrophin-glycoprotein complexes (DGC), the molecular organization from plectin to costameres still remains unclear. Here, we show that plectin 1, a plectin isoform expressed in skeletal muscle, can interact with beta-synemin, actin and a DGC component, alpha-dystrobrevin, in vitro. Ultrastructurally, beta-synemin molecules appear to be incorporated into costameric dense plaques, where they seem to serve as actin-associated proteins rather than IF proteins. In fact, they can bind actin and alpha-dystrobrevin in vitro. Moreover, in vivo immunoprecipitation analyses demonstrated that beta-synemin- and plectin-immune complexes from lysates of muscle light microsomes contained alpha-dystrobrevin, dystrophin, nonmuscle actin, metavinculin, plectin and beta-synemin. These findings suggest a model in which plectin 1 interacts with DGC and integrin complexes directly, or indirectly through nonmuscle actin and beta-synemin within costameres. The DGC and integrin complexes would cooperate to stabilize and fortify the sarcolemma by linking the basement membrane to IFs through plectin 1, beta-synemin and actin. Besides, the two complexes, together with plectin and IFs, might have their own functions as platforms for distinct signal transduction.