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We evaluated the suitability of API 20 STREP and multiplex PCR to speciate 52 Enterococcus spp. obtained from Brazilian foods. A high percentage of isolates (78.9%) presented discrepant results between evaluated tests. Similar results were obtained for six E. faecalis and five E. faecium. The PCR multiplex was more effective than API 20 STREP for complete identification of the isolates. 3633-1936. E-mail: edemarti@usp.br Enterococci are commonly found in soil, water, plants, vegetables and foods (6,9).This genus includes more than 20 species, being E. faecium and E. faecalis the most commonly found (7,8). However, failure in the identification of other species is a matter of concern specially due to the emergence of antibiotic resistant enterococcal strains (12).Identification of enterococci species is generally done by conventional phenotypic and biochemical tests even so molecular methods are becoming the choice tests for bacterial identification (1-3).There are some kits available for a rapid identification of Enterococcus spp. such as ID 32 STREP, API 20 STREP, API 50CH, Api zym (BioMérieux, France) and Phene Plate PhP Plate System (PhPlate Microplate Techniques, Sweden). However, according to Facklam et al. (7) these tests should be carefully used since most of them only correctly identify E. faecalis. The API 20 STREP strip contains 20 microtubes with dehydrated substrates necessary to detect enzymatic activity and fermentation of sugars. The results are obtained within 24 -48 hours, and the identification is based on numerical analysis after a visual interpretation of color changes (BioMérieux, France).The purpose of the present work was to compare the biochemical test -API 20 STREP system and a multiplex Polymerase Chain Reaction (PCR) using specific primers for identification of Enterococcus spp. obtained from Brazilian foods.Fifty two Enterococcus spp. isolates were initialy identified in our lab at genus level based on Gram stain, catalase reaction, growth in de Man Rogosa Sharpe broth -MRS (Oxoid, UK) at 10ºC and 45ºC, MRS broth with 6.5% NaCl, MRS broth pH 9.6 and production of PYR (L-pyrrolidonyl-β-naphthylamide). The presumptive enterococcal isolates were speciated by API 20 STREP (BioMérieux, France), used for identification of streptococci and enterococci, according to the protocol provided by the manufacturer.Multiplex PCR was performed according to protocol described by . Primers were used to amplify the genes ddl of E. faecalis, ddl of E. faecium, vanC-1 of E. gallinarum, vanC-2 and vanC-3 of E. casseliflavus. Overnight cultures of Enterococcus spp. were harvested from trypticase soy agar with 0.6% yeast extract (TSAYE) and transferred to tubes containing 100 µl of PCR water (w-3500-Sigma, USA). This suspension was vortexed, centrifuged for ca. 15 seconds and the supernatant was used as template. The reaction was performed on a DNA thermal cycler (Mastercycler -Eppendorf, Germany) in a final volume of 25 µl containing 60 618 Gomes, B.C. et al.ng of DNA as template, 25 pmol of eac...