An Escherichia coli clinical strain resistant to all beta-lactams except carbapenems was isolated in a Greek hospital. Analysis of beta-lactamase content by isoelectric focusing, PCR assays specific for various bla genes, and DNA sequencing showed that the strain produced TEM-1, a Citrobacter freundii AmpC-related cephalosporinase, and CTX-M-3. The blaCTX.M-3 gene was carried by a 120-kb plasmid that was readily transferable to a susceptible E. coli host.