Localization of Porphyromonas gingivalis and Treponema denticola in different areas of subgingival plaque from advanced adult periodontitis patients was studied immunohistochemically using sensitive immunogold-silver staining and immunoelectron microscopy. Fourteen periodontally diseased teeth were extracted without damaging the subgingival plaque, fixed, and embedded. The subgingival plaque samples were sectioned according to four different pocket depths (0-2 mm, 2-4 mm, 4-6 mm and > or = 6 mm). Serial thin sections were stained using specific antisera to P. gingivalis or T. denticola and then with secondary antibody labelled with colloidal-gold. Cells of both P. gingivalis and T. denticola were predominantly found in subgingival plaque located at depths of more than 4 mm in periodontal pockets. T. denticola cells were found in the surface layers of subgingival plaque, and P. gingivalis were predominant beneath them. However, in the deeper subgingival plaque, the coexistence of P. gingivalis and T. denticola was observed. The present findings suggest that P. gingivalis and T. denticola play important roles in the pathogenicity of periodontal disease and provide the useful information for elucidating the pattern of colonization of microorganisms in the periodontal pocket.