Skeletal muscle contains spectrin (or spectrin I) and fodrin (or spectrin II), members of the spectrin supergene family. We used isoform-specific antibodies and cDNA probes to investigate the molecular forms, developmental expression, and subcellular localization of the spectrins in skeletal muscle of the rat. We report that beta-spectrin (betaI) replaces beta-fodrin (betaII) at the sarcolemma as skeletal muscle fibers develop. As a result, adult muscle fibers contain only alpha-fodrin (alphaII) and the muscle isoform of beta-spectrin (betaISigma2). By contrast, other types of cells present in skeletal muscle tissue, including blood vessels and nerves, contain only alpha- and beta-fodrin. During late embryogenesis and early postnatal development, skeletal muscle fibers contain a previously unknown form of spectrin complex, consisting of alpha-fodrin, beta-fodrin, and the muscle isoform of beta-spectrin. These complexes associate with the sarcolemma to form linear membrane skeletal structures that otherwise resemble the structures found in the adult. Our results suggest that the spectrin-based membrane skeleton of muscle fibers can exist in three distinct states during development.