This study was designed to analyze the succession of an ecosystem of microbial flora in periodontal pockets, in vitro. Bacterial strains used in this study, were Fusobacterium nucleatum ATCC 10953, Porphyromonas gingivalis 381, Streptococcus sanguis ATCC 10557 and Streptococcus mutans OMZ 176. The bacterial cells, associated with beneficial, pathogenic or plaque forming activity, were cultured in brain heart infusion medium including hemin, vitamin-K1, L-cysteine hydrochloride and sodium thioglycollate under anaerobic condition (N2: 80%, CO2: 10%, H2: 10%). The effect of environmental Eh and pH on the growth of bacterial cells was investigated in single and mixed culture, and Eh and pH tolerance tests were also undertaken. The environmental pH decreased from pH 7.0 to pH 4.9 accompanied with the growth of S. mutans OMZ 176 and S. sanguis ATCC 10557 in both single and mixed culture. The environmental Eh increased from -308 mV to -180 mV accompanied with the growth of S. sanguis ATCC 10557. The growth of pathogenic bacteria, such as F. nucleatum ATCC 10953 and P. gingivalis 381, varied markedly with Eh and pH. Especially, the growth of P. gingivalis 381 was severely inhibited at or below pH 6.0 in an acid tolerance test, whereas the growth of F. nucleatum ATCC 10953 was strongly inhibited at Eh -100 mV in an Eh tolerance test. The oxygen generation (10.8%) was confirmed in the anaerobic culture of S. sanguis ATCC 10557. Therefore, it was suggested that hydrogen peroxide produced by S. sanguis ATCC 10557 was reduced to oxygen and water. These results suggest that the high Eh and the low pH generated from bacterial metabolism is a powerful determinant in ecology of microbial flora in periodontal pockets.