After exposure of rat myotube cultures to saponin, less than 1% of the cellular protein was found to remain associated with the tissue culture substrate. This substrate-associated material contained approximately 10% of the acetylcholine receptors (AChRs) and greater than 80% of the large, ventral AChR clusters present in the original culture. The domain structure evident in intact cells was maintained in AChR clusters after isolation using saponin. However, vinculin, present at the clusters of intact cells, was absent from isolated clusters. Dodecyl sulfate PAGE showed that substrate-associated material enriched in AChR clusters contained a distinctive set of polypeptides, the major ones electrophoresing with apparent molecular weights of 43,000 and 49,000. Saponin extraction of cultures of established cell lines also yielded substrate-associated material with characteristics particular to the cell type.