The Cobas-Bact (Roche Diagnostics, Basel, Switzerland) new rotor for the identification (ID) in 4 h 20 min of 33 members of the family Enterobacteriaceae to genus and species level was evaluated by testing 444 strains of which 398 belonged to common species and 46 belonged to rare species of Enterobacteriaceae. Each strain was identified by the API 20E system (Analytab Products, Plainview, N.Y.), and additional discriminating tests were set up if necessary. Only first-choice ID were considered in this study and were classified either as high-confidence ID (normalized likelihood, greater than or equal to 80%) or as low-confidence ID (normalized likelihood, less than 80%) requiring additional tests for confirmation. The data were analyzed by two versions of Cobas-Bact software. With the first version of the software (SW8446), the overall accuracy of Cobas-Bact was 95.5% (424 correct ID of 444). When restricted to high-confidence ID it rose to 99.4% (350 of 352) for the common species and 96.9% (31 of 32) for the rare species. Only three strains of the high-confidence group were misidentified. Sixty ID were considered unacceptable because of their low confidence. Using the first software version (SW8446) they represented 12% (46 of 398) of the common species (17 typical strains, 10 Shigella species, 10 inactive Escherichia coli strains, and 9 rare biotypes) and 30% (14 of 46) of the rare species. The same data analyzed by the new version (SW8524) of the Cobas-Bact software resulted in an overall accuracy of 93.9% (417 correct ID). The number of high-confidence ID rose to 401, of which 392 (97.7%) were accurate. The decrease in low-confidence ID (43 versus 60) was mainly due to the Shigella species. In conclusion the accuracy of Cobas-Bact identification system was very good when restricted to high-confidence ID. The Cobas-Bact performance for rare species ID was poorer, but the small number of strains tested does not allow definitive conclusions.