A novel monoclonal antibody, SM/C-2.6, specific for mouse muscle satellite cells was established. SM/C-2.6 detects mononucleated cells beneath the basal lamina of skeletal muscle, and the cells co-express M-cadherin. Single fiber analyses revealed that M-cadherin⁺ mononucleated cells attaching to muscle fibers are stained with SM/C-2.6. SM/C-2.6⁺ cells, which were freshly purified by FACS from mouse skeletal muscle, became MyoD⁺ in vitro in proliferating medium, and the cells differentiated into desmin⁺ and nuclear-MyoD⁺ myofibers in vitro when placed under differentiation conditions. When the sorted cells were injected into mdx mouse muscles, donor cells differentiated into muscle fibers. Flow cytometric analyses of SM/C-2.6⁺ cells showed that the quiescent satellite cells were c-kit⁻, Sca-1⁻, CD34⁺, and CD45⁻. More, SM/C-2.6⁺ cells were barely included in the side population but in the main population of cells in Hoechst dye efflux assay. These results suggest that SM/C-2.6 identifies and enriches quiescent satellite cells from adult mouse muscle, and that the antibody will be useful as a powerful tool for the characterization of cellular and molecular mechanisms of satellite cell activation and proliferation.