Regulation of the appearance of dihydropyridine-binding sites was studied in primary cultures of chick myotubes. Labeling of surface dihydropyridine-binding sites on intact myotubes at 37 degrees was achieved with (+)-[3H]PN 200-110. The appearance of the sites was prevented in a calcium-free medium using 1.8 mM EGTA, in accordance with the presumed Ca2+ dependency of the appearance of dihydropyridine-binding sites in skeletal muscle cells. Chronic treatment of myotubes with isoproterenol or various other drugs did not modulate either the Bmax or Kd value of the (+)-[3H]PN 200-110 binding to the membrane preparation of treated myotubes (control values were: Kd = 0.16 nm, Bmax = 556 fmol/mg of protein), suggesting a lack of heterologous regulation via beta-adrenergic receptor stimulation. However, depolarization of intact myotubes, either by 47 mM K+ or 10(-5) M veratridine, provoked a 3-fold increase in the Bmax value of (+)-[3H]PN 200-110 binding measured on the intact muscle cells without affecting the Kd value. The effect was reversed upon repolarization of the cells. Depolarizing conditions did not affect the binding on a membrane preparation of the myotubes. Hence, depolarization appeared to specifically trigger the appearance of (+)-[3H]PN 200-110 binding sites on intact myotubes; several hypotheses which could explain the involved mechanism are discussed.