We have investigated whether the phenotype of myogenic clones derived from satellite cells of different muscles from the transgenic immortomouse depended on muscle type origin. Clones derived from neonatal, or 6-to 12-week-old fast and slow muscles, were analyzed for myosin and enolase isoforms as phenotypic markers. All clones derived from slow-oxidative muscles differentiated into myotubes with a preferentially slow contractile phenotype, whereas some clones derived from rapid-glycolytic or neonatal muscles expressed both fast and slow myosin isoforms. Thus, muscle origin appears to bias myosin isoform expression in myotubes. The neonatal clone (WTt) was cultivated in various medium and substrate conditions, allowing us to determine optimized conditions for their differentiation. Matrigel allowed expressions of adult myosin isoforms, and an isozymic switch from embryonic ␣-toward muscle-specific -enolase, never previously observed in vitro. These cells will be a useful model for in vitro studies of muscle fiber maturation and plasticity.