Nerve-evoked contractile activity in skeletal muscle regulates transcript and protein levels of many metabolic genes in a coordinate fashion, including the muscle isozyme of glycogen phosphorylase (MGP). Cellular signaling mechanisms mediating the activity-dependent modulation of MGP transcript levels were investigated in a spontaneously contractile rat skeletal muscle cell line (Rmo). Mechanisms regulating MGP mRNA levels in Rmo myotubes were compared with those previously shown to modulate the gene encoding the β£ subunit of the acetylcholine receptor (β£AChR). Reducing the resting membrane potential from Οͺ78 to Οͺ30 mV, either electrochemically (KCl) or by increasing Na Ο© permeability (veratridine): (1) prevented activation of transverse tubules, (2) impeded calcium release by the sarcoplasmic reticulum (SR), and (3) blocked Rmo contractility. MGP mRNA levels decreased to 30% of control levels and β£AChR levels increased to 350% following 24 h of depolarization. Differing mechanisms appear to mediate this voltage-dependent regulation of MGP and β£AChR. Inhibition of SR calcium efflux selectively decreased MGP mRNA levels by 30 -50% when using dantrolene, thapsigargin, or a dose of ryanodine shown to inactivate Ca 2Ο© -induced SR Ca 2Ο© release (CICR). By contrast, blockade of voltage sensors in transverse tubules with nifedipine, a dihydroaminopyridine (DHAP) antagonist, selectively increased β£AChR mRNA levels by twofold. These data indicate that the voltage-dependent regulation of AChR gene expression differs from that modulating the MGP gene. KCl-induced depolarization and dantrolene both inhibit pulsatile SR Ca 2Ο© efflux in Rmo myotubes, but by differing mechanisms. Depolarization and dantrolene comparably reduced MGP mRNA levels and decreased MGP transcript stability from a t 1/2 of 24 h to 14.5 and 16 h, respectively. Reduced transcript stability can account for the observed reduction in mRNA levels of MGP in noncontractile Rmo myotubes and could be a significant regulatory mechanism in skeletal muscle that coordinates the activity-dependent expression of MGP with other glycogenolytic genes.