The isoprotein-specific intracompartmental sorting of the three essential myosin light chains (LCs), the skeletal muscle LC-1f and LC-3f and the nonmuscle LC-3nm, was investigated. Epitope tagging was used to monitor the intracellular localization to different cytoskeletal structures of the exogenously introduced constructs in adult rat cardiomyocytes (ARCs), which exhibit both stress fibers and regenerating myofibrils. LC-1f and LC-3f bind almost exclusively to the sarcomeric myosin heavy chain (MHC) with high affinity, while the LC-3nm interacts with stress fibers and sarcomeres equally well. Sorting appears to be directed by a hierarchical order of different affinities. Domain mapping by deletion and by construction of a LC-1f/3nm chimera suggests that the LCs are composed of three functionally distinct domains: a basal MHC binding site in the C-terminus; the central part, modulating the preferential interaction with MHC isoforms; and the isoprotein-specific N-terminus of the essential LC, which is probably not involved in the sorting process.