We show that the intermediate filament protein nestin is expressed in myogenic cells and that multiple mechanisms regulate nestin expression at different stages of myogenesis. Cultured embryonic, fetal, and neonatal mouse limb myoblasts initially expressed nestin in the absence of the four muscle regulatory factors (MRFs) of the MyoD family, whereas nestin and MRFs became coexpressed by myoblasts as culture duration was lengthened. Upon differentiation, nestin was commonly concentrated at the ends, and reduced or absent in the middles, of myotubes formed by mouse limb cells. Nestin was expressed by C2C12 and L6 myoblasts and was distributed throughout C2C12 myotubes, but was entirely absent in myotubes formed by L6 cells, suggesting that nestin is dispensable for fusion and terminal differentiation. Nestin was expressed in C3H10T1/2 cells, but was not expressed in 3T3-L1 cells until transfected with MyoD or myogenin. In mouse somites, nestin was found in both myotomal and dermatomal cells. Thus, nestin is expressed by dermatomal cells and by myoblasts during the earliest stages of myogenesis, and nestin expression can be activated upon MRF transfection. Additional MRF-independent mechanisms must, however, regulate nestin expression, because nestin is found in MRF-negative cells and, conversely, nestin is not uniformly distributed in MRF-expressing myotubes.