Beta-lactamases continue to be the leading cause of resistance to beta-lactam antibiotics among gram-negative bacteria. In recent years there has been an increased incidence and prevalence of extended-spectrum beta-lactamases (ESBLs), enzymes that hydrolyze and cause resistance to oxyimino-cephalosporins and aztreonam. The majority of ESBLs are derived from the widespread broad-spectrum beta-lactamases TEM-1 and SHV-1. There are also new families of ESBLs, including the CTX-M and OXA-type enzymes as well as novel, unrelated beta-lactamases. Several different methods for the detection of ESBLs in clinical isolates have been suggested. While each of the tests has merit, none of the tests is able to detect all of the ESBLs encountered. ESBLs have become widespread throughout the world and are now found in a significant percentage of Escherichia coli and Klebsiella pneumoniae strains in certain countries. They have also been found in other Enterobacteriaceae strains and Pseudomonas aeruginosa. Strains expressing these beta-lactamases will present a host of therapeutic challenges as we head into the 21st century.