Objectives: We carried out an evaluation of VITEK 2 in five UK laboratories, comparing results with ‘gold standard’ agar-dilution MIC data, assessing its ability to recognize resistant phenotypes and comparing results with those generated by routine antimicrobial susceptibility testing methods. Methods: Laboratories tested a collection of 82 strains selected on the basis of their challenging and characterized resistance mechanisms. Results: In comparison with the reference MIC method, VITEK 2 gave an essential agreement of 304/315 (enterococci), 1619/1674 (staphylococci) and 2937/3074 (Gram-negative bacilli): overall 96.0% agreement. Corresponding category (SIR) agreements with VITEK 2 were 247/252, 1496/1561 and 2478/2626 (overall 95.1%). Using five routine methodologies, category agreements ranged from 58/63 to 45/45; 222/232 to 174/174, and 333/372 to 250/259 for the three organism groups with an overall agreement of 95.0%. In contrast to VITEK 2 Advanced Expert System (AES), routine microbiology laboratories did not attempt to detect resistance mechanisms for every antibiotic studied. VITEK 2 AES detected all 19 resistance mechanisms in enterococci: where applicable, routine methods detected 14, 10 and 10. Of 30 resistance mechanisms in staphylococci, VITEK 2 AES detected 25 compared with 23, 20, 17 and 18 detected by routine methods. Finally, of 44 resistance mechanisms in Gram-negative bacilli, VITEK 2 AES detected 30 compared with 30, 23, 15 and 10 detected by routine methods. Conclusions: VITEK 2 performed susceptibility tests accurately and the AES detected and interpreted resistance mechanisms appropriately. Heavy inocula in a liquid medium possibly favour better expression of certain resistance determinants. Although certain routine microbiology methods performed adequately, VITEK 2 AES offers a rapid, standardized method suited to laboratories lacking experience of resistance mechanisms and/or those not testing an appropriate number, or range, of antibiotics to detect resistance phenotypes.