Abstract. Satellite cells were isolated at high yields from slow-twitch soleus and fast-twitch tibialis anterior (TA) muscles of adult male Wistar rats. The number of satellite cells isolated from soleus muscle exceeded that from TA muscles by a factor of three. A comparison of satellite cells grown on gelatin- or Matrigel-coated dishes revealed that Matrigel greatly enhances the maturation of the satellite-cell-derived myotubes. As judged from immunohistochemistry, myosin heavy chain electrophoresis and immunoblot analyses, only cells grown on Matrigel, but not on gelatin, expressed adult myosin isoforms. Slow myosin expression was only detected in Matrigel cultures. Soleus cultures contained, in addition to the majority of myotubes expressing fast myosin, a small fraction (maximally 10%) of myotubes coexpressing fast and slow myosins. The number of fast/slow myosin-containing myotubes was negligible in TA cultures. The expression of slow myosin increased with age. Slow myosin was nonuniformly distributed along the length of specific myotubes and accumulated around some myonuclei. These results point to the existence of myotubes with a heterogeneous population of myonuclei, probably resulting from fusion of differently preprogrammed satellite cells. We suggest that the patch-like expression of slow myosin results from local accumulation of myonuclei of slow-type satellite cells.