The development and turnover of acetylcholine receptors in a nonfusing muscle cell line has been investigated using iodinated alpha-bungarotoxin as a probe for acetylcholine receptor. logarithmically growing cells do not bind toxin, while cells that have ceased cell division bind toxin at a site which has the pharmacological characteristics of an acetylcholine receptor. These binding sites are removed from the cell surface at a rate equal to 8.9 +/- 0.5% of the total surface binding sites/h and appear at a rate equal to 8.3 +/- 1.5% of the total surface binding sites/h. Appearance of new binding sites can occur for a period of 1 1/2 h in the presence of cycloheximide, during which time 15% of the surface receptors can be replaced. There is a hidden population of receptors which is not accessible to toxin without disrupting the cell. This population amounts to 35% of the Triton-extractable receptors in the cell and is composed of two classes. One class, termed a precursor receptor, appears to move from the hidden population to the cell surface, and composes about 40% of the total hidden receptor population. The second class of hidden receptors does not appear to function as a surface precursor and is neither depleted nor enriched by any of the procedures we employed. Surface receptors and hidden receptors are distinguishable on the basis of their sedimentation coefficient about 0.5 to 0.6 S lower than surface receptors. We were unable to distinguish between precursor and non-precursor hidden receptors on the basis of cursor and nonprecursor hidden receptors on the basis of their sedimentation coefficients.