Extended-spectrum β-lactamases (ESBLs) are plasmid-mediated bacterial enzymes that confer resistance to a broad range of β-lactams. They are descended by genetic mutation from native β-lactamases found in gram-negative bacteria, especially infectious strains of Escherichia coli and Klebsiella species. Genetic sequence modifications have broadened the substrate specificity of the enzymes to include third-generation cephalosporins, such as ceftazidime. Because ESBL-producing strains are resistant to a wide variety of commonly used antimicrobials, their proliferation poses a serious global health concern that has complicated treatment strategies for a growing number of hospitalized patients. Another resistance mechanism, also common to Enterobacteriaceae, results from the overproduction of chromosomal or plasmid-derived AmpC β-lactamases. These organisms share an antimicrobial resistance pattern similar to that of ESBL-producing organisms, with the prominent exception that, unlike most ESBLs, AmpC enzymes are not inhibited by clavulanate and similar β-lactamase inhibitors. Recent technological improvements in testing and in the development of uniform standards for both ESBL detection and confirmatory testing promise to make accurate identification of ESBL-producing organisms more accessible to clinical laboratories.