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The persistent presence of bacteria in the root canal system often leads to the failure of treatment. The purpose of this study was to determine in vitro penetration of Streptococcus sanguis and Prevotella intermedia into dentinal tubules. Samples obtained from human teeth were inoculated with a strain of S. sanguis (NCTC 7853) and P. intermedia (NCTC 93336) for 20 days. Bacterial penetration into tubules was investigated at scanning electron microscopy and light microscopic level. The results showed that S. sanguis could penetrate into dentinal tubules 382.3 μm, whereas P. intermedia could penetrate 25.9 μm. It was observed that P. intermedia had not penetrated into all dentinal tubules. If penetration occurred the depth was quite limited.