In electron microscopy, the post-embedding immunogold technique provides a high degree of resolution and the possibility of quantitation owing to the intrinsic characteristics of the colloidal gold marker. Application of this technique to the subcellular localization of multiple antigens by differential labeling using gold markers of different sizes, or to double labeling using the same primary antibody isotype with serial silver enhancement, has been reported. We have incorporated this double labeling technique into a modified procedure that produces excellent labeling and ultrastructural preservation, even after exposure of ultrathin sections large enough to cover a 300- micro m-diameter single-hole grid to hot antigen retrieval solutions and prolonged labeling protocols.