The relation among sodium channel density, frequency of electrical activity and maximal rate of rise of the action potential was studied in developing and mature rat skeletal myotubes in culture. The number of tetrodotoxin (TTX)-sensitive Na-channels was determined by measurements of the amount of [3H]saxitoxin (STX) bound to the cultures, and electrical properties were recorded with intracellular microelectrodes. The EC50 for TTX-induced decreases in maximal STX-binding, frequency and rate of rise of action potentials was in the range 8-20 nM. The 3 variables increased in parallel with age in culture to reach peak values at age 7-8 days, and then decreased in parallel until 10-12 days in culture. The age-related increase in Na-channel density was decreased, but not abolished, by prevention of myoblast fusion. Treatment with the Ca2+ ionophore, A23187, down-regulated, and blockade of Ca-channels with verapamil up-regulated the number of Na-channels. Na-channel density was also increased by chronic treatment with TTX and elevated external [K+], which eliminated spontaneous electrical and contractile activity. Parallel effects were observed on frequency and rate of rise of action potentials. Up-regulation of Na-channels was prevented by simultaneous treatment of myotubes with inhibitors of protein synthesis. We conclude that electrical and mechanical activity of cultured myotubes regulate de novo synthesis of Na-channels through alterations in the level of cytosolic Ca2+.