This study analyzes changes in the distribution, electrophysiological propertiesand proteic composition of voltage-gated sodium channels (Na(V)) in cultured adult rat skeletal muscle fibers. Patch clamp and molecular biology techniques were carried out in flexor digitorum brevis (FDB) adult rat skeletal muscle fibers maintained in vitro after cell dissociation with collagenase. After 4 days of culture, an increase of the Na(V)1.5 channel type was observed. This was confirmed by an increase in TTX-resistant channels and by Western blot test. These channels exhibited increased activation time constant (tau(m)) and reduced conductance, similar to what has been observed in denervated muscles in vivo, where the density of Na(V)1.5 was increasing progressively after denervation. By real-time polymerase chain reaction, we found that the expression of beta subunits was also modified, but only after 7 days of culture: increase in beta(1) without beta(4) modifications. beta(1) subunit is known to induce a negative shift of the inactivation curve, thus reducing current amplitude and duration. At day 7, tau(h) was back to normal and tau(m) still increased, in agreement with a decrease in sodium current and conductance at day 4 and normalization at day 7. Our model is a useful tool to study the effects of denervation in adult muscle fibers in vitro and the expression of sodium channels. Our data evidenced an increase in Na(V)1.5 channels and the involvement of beta subunits in the regulation of sodium current and fiber excitability.