The purpose of the present investigation was to compare the microbial composition of supra and subgingival plaque in 22 periodontally healthy (mean age 32+/-16 years) and 23 adult periodontitis subjects (mean age 51+/-14 years). A total of 2358 supra and separately subgingival plaque samples were collected from the mesial aspect of all teeth excluding 3rd molars in each subject. Samples were examined 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. Mean counts (x10(5), % DNA probe count and % sites colonized for each species were determined separately for supra and subgingival samples in each subject and then averaged across subjects in the 2 clinical groups. Significance of differences between healthy and periodontitis subjects was determined using the Mann-Whitney test and adjusted for multiple comparisons. Mean total DNA probe counts (x10(5), +/-SEM) for healthy and periodontitis subjects in supragingival plaque were 72.1+/-11 and 132+/-17.5, respectively (p<0.01), and in subgingival plaque 22.1+/-6.6 and 100.3+/-18.4, (p<0.001). Porphyromonas gingivalis, Bacteroides forsythus and Treponema denticola could be detected in supragingival plaque samples of both healthy and periodontitis subjects. Actinomyces species were the dominant taxa in both supra- and subgingival plaque from healthy and periodontitis subjects. 4 Actinomyces species accounted for 63.2%, of supragingival and 47.2% of subgingival plaque in healthy subjects and 48.% and 37.8% in periodontitis subjects respectively. Increased proportions of P. gingivalis, B. forsythus, and species of Prevotella, Fusobacterium, Campylobacter and Treponema were detected subgingivally in the periodontitis subjects. P. gingivalis, B. forsythus and T. denticola were significantly more prevalent in both supra- and subgingival plaque samples from periodontitis subjects. The main differences between supra and subgingival plaque as well as between health and disease were in the proportions and to some extent levels of Actinomyces, "orange" and "red" complex species.