OBJECTIVE: To evaluate which of 24 beta-lactams used in susceptibility tests best discriminated between strains of Klebsiella pneumoniae and Escherichia coli that produce extended spectrum beta-lactamases (ESBLs) from strains that produce older, more familiar, plasmid-mediated beta-lactamases such as TEM-1 and SHV-1. METHODS: Susceptibility to the 24 beta-lactam agents was determined by agar dilution and disk diffusion methodologies, using 27 strains of K. pneumoniae and E. coli that produced 22 different older plasmid-mediated beta-lactamases and 28 strains that produced 17 different ESBLs. RESULTS: In general, strains that produced ESBLs were intermediate or resistant to cefpodoxime, whereas those that produced other beta-lactamases were susceptible to this agent. The agar dilution test exhibited 96% sensitivity and 100% specificity in discriminating these two groups of organisms. The disk diffusion test exhibited 100% sensitivity and 96% specificity. All other beta-lactam agents tested were inferior discriminators between the two groups of organisms. CONCLUSIONS: Agar dilution and disk diffusion tests with cefpodoxime can be used to discriminate strains of K. pneumoniae and E. coli that produce ESBLs from those that produce older, plasmid-mediated beta-lactamases.