Background: Enterococcci were found to be associated with failed endodontic treatment by traditional methods. Commercial kits are often used by clinical laboratories to identify enterococcal species. However, some studies have shown discordance between the results of the commercial kits and the molecular identification methods. Objectives: The aim of the present study was to speciate Enterococcus strains isolated in Brazil from 60 canals of root-filled teeth with periapical lesions using biochemical and molecular techniques. Methods: Twenty-one Enterococcus strains were identified to the species level using Rapid ID 32 STREP galleries. Genotypic identification was performed by 16S rDNA sequencing: 16S rDNA amplification was carried out with 2 universal primers (RE-TPU1 and RE-RTU3), the PCR products were sequenced by dye-terminator sequencing, and the sequences were analyzes by the BLAST software of the National Centre for Biotechnology Information. Results: All strains were identified as Enterococcus faecalis by both 16S rDNA sequencing and the commercial kit, which gave high probabilities of correct identification (>99% and >90% respectively). Conclusion: Enterococcus faecalis was the most common enterococcal species isolated from canals of root-filled teeth with periapical lesions. Strain differentiation will require further studies using Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing. (Supported by the Brazilian agencies FAPESP 00/13686-8, 00/13689-7, CAPES BEX2449/02-1 and CNPq -520277/99-6).

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