In skeletal muscle, the cytolinker plectin is prominently expressed at Z-disks and the sarcolemma. Alternative splicing of plectin transcripts gives rise to more than eight protein isoforms differing only in small N-terminal sequences (5–180 residues), four of which (plectins 1, 1b, 1d, and 1f) are found at substantial levels in muscle tissue. Using plectin isoform–specific antibodies and isoform expression constructs, we show the differential regulation of plectin isoforms during myotube differentiation and their localization to different compartments of muscle fibers, identifying plectins 1 and 1f as sarcolemma-associated isoforms, whereas plectin 1d localizes exclusively to Z-disks. Coimmunoprecipitation and in vitro binding assays using recombinant protein fragments revealed the direct binding of plectin to dystrophin (utrophin) and β-dystroglycan, the key components of the dystrophin–glycoprotein complex. We propose a model in which plectin acts as a universal mediator of desmin intermediate filament anchorage at the sarcolemma and Z-disks. It also explains the plectin phenotype observed in dystrophic skeletal muscle of mdx mice and Duchenne muscular dystrophy patients.