Background, aims: The purpose of the present study was to compare and relate the microbial composition of supra and subgingival plaque in 23 adult periodontitis subjects (mean age 51±14 years).Methods: A total of 1,170 samples of supra and subgingival plaque were collected from the mesial aspect of every tooth (up to 28 supra and 28 subgingival samples) from each subject and evaluated for the presence and levels of 40 bacterial taxa using whole genomic DNA probes and checkerboard DNA‐DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. The counts (levels), % DNA probe count (proportion) and % of sites colonized (prevalence) of each species in supra and separately in subgingival plaque were computed for each subject. Significance of differences between supra and subgingival plaque for each species was sought using the Wilcoxon signed ranks test and adjusted for multiple comparisons.Results: All 40 taxa were detected in both supra and subgingival plaque. Actinomyces species were the most prevalent taxa in both habitats. 75 to 100% of supra and 62 to 100% of subgingival sites were colonized by at least one of the 5 Actinomyces species. Supragingival samples exhibited significantly higher counts of Actinomyces naeslundii genospecies 1, Actinomyces israelii, Actinomyces odontolyticus, Neisseria mucosa, Streptococcus gordonii, Capnocytophaga ochracea and Capnocytophaga sputigena when compared with mean counts in subgingival samples taken from the same tooth surfaces. Subgingival plaque samples presented significantly higher counts of Prevotella nigrescens, Prevotella intermedia, Bacteroides forsythus and Porphyromonas gingivalis. Subgingival samples exhibited a significantly higher proportion of “red” and “orange complex” species, while supragingival plaque exhibited higher proportions of “green” and “purple” complex species as well as Actinomyces species. Suspected periodontal pathogens could be detected in supragingival plaque from sites where subgingival samples were negative for the same species.Conclusions: The data indicate that supragingival plaque can harbor putative periodontal pathogens, suggesting a possible rôle of this environment as a reservoir of such species for the spread or reinfection of subgingival sites.