Membrane depolarization inactivates acetylcholine receptor (AChR) genes in skeletal muscle. We have studied this process in C2C12 cells, focusing on the role of calcium. Cytoplasmic calcium was monitored with fluo-3, and the activity of receptor genes was measured with a sensitive transcript elongation assay. Removal of extracellular calcium or blockage of L-type calcium channels disrupts signaling, even when release of calcium from the sarcoplasmic reticulum (SR) is not impeded, whereas L channel agonists induce signaling without membrane depolarization or release of calcium from intracellular stores. Activators of calcium release from the SR do not inhibit AChR genes, either in C2C12 or in chicken skeletal muscle in vivo. It appears that calcium ions do not act as messengers between sarcolemma and nucleus but target a sensor near their port of entry where they initiate a signal that bypasses the SR.