A semi-automated commercial system (ID 32 E, bioMérieux) for 24-hour identification of Enterobacteriaceae and other gram-negative fermentative and nonfermentative bacteria encountered in diagnostic microbiology was evaluated. Overall, the system correctly identified 506 (91.5%) of the 553 strains tested, 94 (17.0%) strains requiring additional tests for complete identification. Six (1.1%) strains were misidentified and 33 (6.0%) strains were not identified. Eight (1.4%) strains were not present in the database and were misidentified or not identified. The system is a suitable alternative to existing systems for the identification of Enterobacteriaceae and other gram-negative bacteria frequently encountered in clinical samples.