From replicate trials of experimental gingivitis in four periodontally healthy subjects, 166 bacterial species and subspecies were detected among 3,034 randomly selected isolates from 96 samples. Of these bacteria, Actinomyces naeslundii (serotype III and phenotypically similar strains that were unreactive with available antisera), Actinomyces odontolyticus (serotype I and phenotypically similar strains that were unreactive with available antisera), Fusobacterium nucleatum, Lactobacillus species D-2, Streptococcus anginosus, Veillonella parvula, and Treponema species A appeared to be the most likely etiological agents of gingivitis. Statistical interpretations indicated that the greatest source of microbiological variation of the total flora observed was person-to-person differences in the floras. The next greatest source of variation was the inflammatory status of the sample sites. Person-to-person differences were smallest at experimental day 4. The floras became more diverse with time and as gingivitis developed and progressed. Analyses indicated that sequential colonization by certain species was repeatable and therefore probably predictable. Variation was relatively small between replicate trials, between two sites on the same teeth sampled on the same day, and between the same sites sampled at the same relative time in a replicate trial.