This investigation included a cross-sectional component (284, 20-40-year-old subjects/568 sites) and a longitudinal component (19 subjects with moderate to advanced periodontitis/76 sites). Subgingival plaque samples and clinical data were obtained from all upper second bicuspids and first molars in the longitudinal subjects and from both upper first molars in the cross-sectional subjects. The purpose was to: determine/confirm changes in proportions of selected subgingival microorganisms following root planning combined with conventional oral hygiene procedures in the longitudinal subjects, evaluate those changes in terms of an estimate of the naturally occurring distribution of subgingival microorganisms based on data obtained from the cross-sectional subjects and relate shifts in the subgingival microorganisms to changes in clinical measurements. Changes following treatment at sites with pre-instrumentation probing depths greater than or equal to 4.5 mm included cocci, 18.3 to 46.9%; spirochetes, 20.9 to 3.1%; total motile organisms, 28.5 to 5.9%; Fusobacterium spp., 10.2 to 2.2%; and dark-pigmented Bacteroides spp., 14.0 to 9.7%. Translating proportions of microorganisms in samples from subjects in the longitudinal treatment study to equivalent percentile ranks within the large cross-sectional data base demonstrated that at probing depths greater than or equal to 4.5 mm cocci moved from below the 50th percentile in our estimate of the naturally occurring distribution of subgingival microorganisms to above, and spirochetes, total motile organisms and Fusobacterium spp. moved from above the 50th percentile to below. The microbiological findings were consistent with statistically significant improvements in clinical measurements. Statistically significant changes also occurred in microorganisms at sites with initial probing depths less than 4.5 mm.