We have examined the relationship of acetylcholine receptors (AChR) to the Mr 43,000 receptor-associated protein (43K) in the AChR clusters of cultured rat myotubes. Indirect immunofluorescence revealed that the 43K protein was concentrated at the AChR domains of the receptor clusters in intact rat myotubes, in myotube fragments, and in clusters that had been purified approximately 100-fold by extraction with saponin. The association of the 43K protein with clustered AChR was not affected by buffers of high or low ionic strength, by alkaline pHs up to 10, or by chymotrypsin at 10 micrograms/ml. However, the 43K protein was removed from clusters with lithium diiodosalicylate or at alkaline pH (greater than 10). Upon extraction of 43K, several changes were observed in the AChR population. Receptors redistributed in the plane of the muscle membrane in alkali-extracted samples. The number of binding sites accessible to an anti-AChR monoclonal antibody directed against cytoplasmic epitopes (88B) doubled. Receptors became more susceptible to digestion by chymotrypsin, which destroyed the binding sites for the 88B antibody only after 43K was extracted. These results suggest that in isolated AChR clusters the 43K protein covers part of the cytoplasmic domain of AChR and may contribute to the unique distribution of this membrane protein.