In this study, the membrane potential and cytosolic [Ca2+] were measured in rat myotubes developing in culture from days 6-14. It was found that as the myotubes developed in culture, the resting membrane potential (RMP) became more negative during days 6-8, and then did not significantly change until after day 13, when it started to become less negative. The mean RMP measured at days 8-13 was -59 +/- 1 mV (n = 70). The amplitude of action potentials elicited in the myotubes by anode break stimulation increased in size during development (range: 47.5-119 mV) and this closely correlated with the development of a more negative RMP. Cytosolic [Ca2+] was measured in the rat myotubes using the Ca2+ indicator Fura-2, and no significant change in the resting [Ca2+] was observed during development (days 6-14). Ca2+ responses triggered by action potentials varied from small slow increases in [Ca2+] that failed to return to the baseline to rapid [Ca2+] transients. The size of the [Ca2+] transients positively correlated with both the observed increase in the RMP during development and the size of the action potential. Larger [Ca2+] transients also had more rapid rates of [Ca2+] decay, indicating a tandem increase in the ability of the sarcoplasmic reticulum to release and resequester Ca2+ during development of rat myotubes. Repetitive stimulation (10 Hz) of the myotubes exhibiting small [Ca2+] transients produced a step-like rise in [Ca2+]. Many myotubes exhibiting larger [Ca2+]transients could not be stimulated at 10 Hz by anode break stimulation due to the presence of action potentials with large hyperpolarisations. However, when these myotubes were depolarised at 10 Hz, they produced a tetanic Ca2+ response similar to that seen in adult skeletal muscle.