Mononucleated myogenic cells (satellite cells) were isolated from skeletal muscle of adult rats and grown in culture. These cells replicated and, beginning with the 6th day in culture, they fused and differentiated into multinucleated myotubes, which accumulated creatine kinase and developed cross striation and spontaneous contractions. The differentiation of the excitable membrane and the action of sea anemone toxin ATX II were investigated with microelectrode techniques. Mature myotubes reached a stable membrane potential of -47.3 mV (+/- 6.5 mV) with the 11th day in culture. Action potentials could be generated in all myotubes. During maturation they became faster (increasing rate of rise) and shorter in duration. In spontaneously contracting myotubes spontaneous action potentials were recorded, which were often associated with subthreshold oscillations of membrane potential. ATX II reduced the membrane potential and prolonged the action potential duration with the lowest effective concentrations being 1 nmol/l and 0.5 nmol/l, respectively. Furthermore, ATX II induced electrical activity in quiescent myotubes. After fusion the development of the membrane electrical properties of satellite cell derived muscle cells followed essentially the same pattern as in primary cultures of embryonic myotubes. Electrophysiologically and with respect to their sensitivity to ATX II the mature myotubes resemble denervated muscle fibres.