The level of cytoplasmic calcium has been proposed to act as a regulator of acetylcholine receptor synthesis (Betz, H., and J. P. Changeaux (1979) Nature 278: 749–751). However, there is little known about the effect of altered calcium levels on the metabolism of the acetylcholine receptor. We have investigated the effect of decreased extracellular calcium on the metabolism of acetylcholine receptors in cultured rat myotubes. Our results show that the acetylcholine receptor levels on the surface of myotubes were decreased 25 to 30% following overnight incubation in calcium-deficient medium. In contrast, creatine phosphokinase activity levels and total protein synthesis were unaffected. Calcium depletion did not change the rate of receptor degradation significantly (0.037 hr-1, compared to 0.033 hr-1 for control cells) but dramatically decreased the rate of incorporation of new acetylcholine receptors into the plasma membrane. The time course for incorporation of new acetylcholine receptors into the plasma membrane of calcium-depleted cells was similar to control cells treated with cycloheximide, suggesting that de novo receptor synthesis was inhibited. These results indicate that intracellular calcium levels and acetylcholine receptor synthesis are not related in a simple reciprocal fashion and suggest that the regulation of acetylcholine receptor levels involves more than one intracellular compartment of calcium.