The human erythrocyte membrane skeleton consists of a network of short actin filaments cross-linked into a hexagonal network by long, flexible spectrin molecules. The lengths of the short actin filaments (33 +/- 5 nm) at the central junctions are proposed to be stabilized and limited by association with tropomyosin and the tropomyosin-binding protein, tropomodulin. Here, we use immunogold labelling followed by negative staining to specifically localize tropomodulin, tropomyosin and actin to the sites of the central junctions in spread membrane skeletons. In addition to negative staining, immunogold labelling for tropomodulin at the sites of the central junctions was also visualized by a quick-freeze, deep-etch, rotary-replication technique. These experiments confirm previous indirect evidence that the short filaments at the central junctions are indeed actin filaments and provide the first direct evidence that tropomodulin and tropomyosin are associated with the erythrocyte actin filaments in situ.