We modified the lipids of rat myotubes in tissue culture to determine the transmembrane orientation of aminophospholipids in clusters of acetylcholine receptors (AChR). Trinitrobenzenesulfonic acid and N-hydroxysuccinimidobiotin were used to modify the amino groups of phospholipids. Reaction conditions were selected to prevent penetration of the chemical probes into the cell interior. Fluorescence microscopy was used to confirm that the probes remained impermeant. Analysis of aminophospholipids associated with clusters isolated from chemically modified cells and comparisons to results of chemically modifying isolated acetylcholine receptor clusters indicated that at least 77% of plasma membrane aminophospholipids was located in the interior leaflet of the lipid bilayer. We address the possibility that aminophospholipids on the inner lipid leaflet may contribute to the association between the cytoskeleton and the membrane at AChR clusters.