Mouse skeletal myotubes differentiated in vitro exhibited spontaneous contractions associated with electrical activity. The ionic conductances responsible for the origin and modulation of the spontaneous activity were examined using the whole-cell patch-clamp technique and measuring [Ca(2+)](i) transients with the Ca(2+) indicator, fura 2-AM. Regular spontaneous activity was characterized by single TTX-sensitive action potentials, followed by transient increases in [Ca(2+)](i). Since the bath-application of Cd(2+) (300 microM) or Ni(2+) (50 muM) abolished the cell firing, T-type (I(Ca,T)) and L-type (I(Ca,L)) Ca(2+) currents were investigated in spontaneously contracting myotubes. The low activation threshold (around -60 mV) and the high density of I(Ca,T) observed in contracting myotubes suggested that I(Ca,T) initiated action potential firing, by bringing cells to the firing threshold. The results also suggested that the activity of I(Ca,L) could sustain the [Ca(2+)](i) transients associated with the action potential, leading to the activation of apamin-sensitive SK-type Ca(2+)-activated K(+) channels and the afterhyperpolarization (AHP) following single spikes. In conclusion, an interplay between voltage-dependent inward (Na(+) and Ca(2+)) and outward (SK) conductances is proposed to mediate the spontaneous pacemaker activity in cultured muscle myotubes during the process of myogenesis.