Voltage-gated sodium channels (Na(v)) consist of a pore-forming alpha subunit (Na(v)alpha) associated with beta regulatory subunits (Na(v)beta). Adult skeletal myocytes primarily express Na(v)1.4 channels. We found, however, using neonatal L6E9 myocytes, that myofibers acquire a Na(v)1.5-cardiac-like phenotype efficiently. Differentiated myotubes elicited faster Na(v)1.5 currents than those recorded from myoblasts. Unlike myoblasts, I(Na) recorded in myotubes exhibited an accumulation of inactivation after the application of trains of pulses, due to a slower recovery from inactivation. Since Na(v)beta subunits modulate channel gating and pharmacology, the goal of the present work was to study Na(v)beta subunits during myogenesis. All four Na(v)beta (Na(v)beta1-4) isoforms were present in L6E9 myocytes. While Na(v)beta1-3 subunits were up-regulated by myogenesis, Na(v)beta4 subunits were not. These results show that Na(v)beta genes are strongly regulated during muscle differentiation and further support a physiological role for voltage-gated Na(+) channels during development and myotube formation.