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The M-band is the transverse structure that cross-links the thick filaments in the center and provides a perfect alignment of the A-band in the activated sarcomere. The molecular composition of the M-bands in adult mouse skeletal muscle is fiber-type dependent. All M-bands in fast fibers contain M-protein while M-bands in slow fibers contain a significant proportion of the EH-myomesin isoform, previously detected only in embryonic heart muscle. This fiber-type specificity develops during the first postnatal weeks. However, the ratio between the amounts of myosin and of myomesin, taken as sum of both isoforms, remains nearly constant in all studied muscles. Ultrastructural analysis demonstrates that some of the soleus fibers show a diffuse appearance of the M-band, resembling the situation in the embryonic heart. A model is proposed to explain the functional consequence of differential M-band composition for the physiological and morphological properties of sarcomeres in different muscle types.