The regulation of the number of extrajunctional acetylcholine (ACh) receptors was assayed by 125I-labelled alpha-bungarotoxin binding sites in denervated rat diaphragm muscle in culture. Sustained K depolarization does not eliminate extrajunctional ACh receptors. In fact, muscle cultured in high-K medium (normal Cl) for 3 days exhibits a greater binding capacity than controls. Under conditions in which the intracellular Cl concentration is unaltered (high-K-low-Cl medium) this effect of high-K medium on the number of extra-junctional ACh receptors is blocked. The number of extrajunctional receptors increases 24-48 h after exposure to high-K-normal Cl medium, similar to the time course of the initial appearance of extrajunctional receptors in the denervated diaphragm muscle in vivo or in organ culture in normal media. High-K-normal Cl medium did not alter the rate of receptor degradation. Electrical stimulation of denervated muscle strips cultured in low-Ca medium containing D-600 eliminated extrajunctional receptors as efficiently as stimulation of muscles in control medium. Electrical stimulation did not reduce the extrajunctional ACh receptor population in glycerol-treated uncoupled muscles to the same extent as in untreated muscles. The extrajunctional ACh receptor content of denervated muscle cultured for 3 days in 2 and 5 mM-caffeine was reduced by about half respectively. Denervated muscle cultured in 0.3 mM-caffeine did not differ from control denervated muscle. Other agents which may alter intracellular cyclic nucleotide levels: dibutyryl cyclic GMP, dibutyryl cyclic AMP, papaverine, and sodium nitroprusside, did not mimic the effect of caffeine or electrical stimulation in lowering the levels of extrajunctional ACh receptors. We conclude that intracellular Ca release from the sarcoplasmic reticulum is necessary for the elimination of extrajunctional ACh receptors in denervated muscle. The levels of intracellular Cl also influence the population of extrajunctional receptors. Conditions which lead to higher levels of intracellular Cl result in greater rates of synthesis of ACh receptors.