Cultured neonatal rat myotubes develop acetylcholine receptor (AChR) clusters where they adhere to the substrate. These clusters are often linear, with AChR-rich domains alternating with AChR-poor "contact domains" closer to the tissue culture substrate. We have used sequential detergent extraction and immunofluorescence microscopy to localize extracellular matrix components within these two domains. Laminin, heparan sulfate proteoglycan, type IV collagen, and fibronectin are present at AChR-rich domains; nerve cell adhesion molecule is present at both AChR and contact domains. Extracts of contact domains are enriched in a 36-kDa concanavalin A binding protein and in a 90-kDa polypeptide recognized by antibodies against rat muscle adherons. These results suggest that extracellular components at substrate-apposed AChR clusters are organized into distinct domains that parallel the organization of the cluster bilayer.