OBJECTIVES The aim of this study was to evaluate phenotypic detection of beta-lactamase-mediated resistance to oxyimino-cephalosporins in Enterobacteriaceae using the Mastascan Elite Expert System challenged with a battery of genotypically characterized organisms. METHODS Isolates (n = 120) were identified to species level and antimicrobial susceptibilities were determined using agar incorporation methods and Mastascan Elite. Phenotypes were examined using an Expert System (ES) and putative genotypes were suggested using interpretative reading. RESULTS Identification was correct in 119 of 120 isolates. The ES was able to identify the correct beta-lactam phenotype (as deduced from molecular methods) in a single choice in 98 of 120 (81.7%) isolates. In an additional 15 (12.5%) cases, the ES identified the correct beta-lactam phenotype within two or more choices. The detected phenotype was incorrect in seven (5.8%) isolates, but three of these were not inherent to the ES. CONCLUSIONS The Mastascan Elite ES is relatively inexpensive and flexible and can identify the mechanism of resistance to oxyimino-cephalosporins in the majority of Enterobacteriaceae without recourse to molecular methods.