Acquisition of acetylcholine receptors during differentiation of a clonal myoblast cell line was monitored with a neurotoxin isolated from venom of the Indian Cobra Naja naja. Toxin bound specifically and reversibly to acetylcholine receptors of the differentiated cells. Specificity of the binding reaction was assayed by measurement of the ability of various cholinergic agonists and antagonists to compete with neurotoxin for its binding site. The rate of toxin binding paralleled the rate of inactivation of functional acetylcholine receptors, as measured by iontophoretic application of acetylcholine. Bound toxin was released from the cells with a half-life of about 7 hr. This release was not associated with a decrease in the total number of toxin-binding sites. A slow hyperpolarizing response to acetylcholine seen in myoblasts was insensitive to toxin; the appearance of toxin-binding sites parallels the appearance of fused fibers during differentiation of the muscle cells in tissue culture.