The localization and distribution of Porphyromonas gingivalis, Campylobacter rectus and Actinomyces viscosus were studied in human periodontal pockets. After obtaining voluntary consent from 9 patients, 12 teeth and their surrounding periodontal tissue with advanced adult periodontitis were extracted carefully so as not to change the structure of the periodontal pockets. The specimens were processed into serial sections. One of the sections was stained with Brown & Brenn-modified Gram stain to observe the distribution of bacteria. The others were stained immunohistochemically by the Labelled Streptavidin Biotin method (LSAB method) using specific rabbit antibodies against selected bacteria. Some bacteria could be found within epithelial cells. P. gingivalis was found in 9/12 of the samples examined. Small aggregates of P. gingivalis were scattered in all parts of the periodontal pockets, and some of these aggregates could be seen in close contact with the epithelium. Conversely, C. rectus was observed in 5/12 of the samples examined and was predominantly located in the middle and deep pocket zones. C. rectus tended to form large clumps in both the tooth-attached and epithelium-associated plaque area. A. viscosus was observed in 7/12 of the samples examined and was localized predominantly in the tooth-attached plaque area, especially in the shallow and middle pocket zones. Although unexpected spills of unattached plaque from periodontal pockets was possible, immunohistochemical staining with species-specific antibodies was extremely sensitive and revealed the localization and the distribution of periodontal disease-associated bacteria in human periodontal pockets.