Electrophysiological and autoradiographic studies have shown that the distribution of acetylcholine (AcCho) receptors on uninnervated cultured chicken muscle cells is not uniform. Regions of high receptor density (hot spots 10--40 times more sensitive than surrounding areas are localized as discrete patches or clusters about 10 micrometer in diameter o myotube muscle membranes. Hot spots were also found on fusion-arrested mononucleated myoblasts. We have developed a method for freeze-fracturing monolayer cultures that allows the unambiguous reidentification of membrane patches previously assayed for ACCho sensitivity. The freeze-fractured membranes at physiologically defined hot spots contain aggregates of many (10--20) small clusters of large (10--19 nm in diameter) intramembranous particles. Clusters are found on both fracture faces, but the particle density is much greater on the protoplasmic (P) face than on the extracellular (E) face (about 2000/micrometer2 vs. 700/micrometer2). Some of the particles appear to be composed of five or six "subunits" arranged cylindrically around a central dark dot. Because the aggregates are present at sites of high AcCho sensitivity, it is likely that the intramembranous particles are in some way related to the AcCho receptor molecule.