Extended-broad-spectrum beta-lactamase TEM-9, detected in a clinical isolate of Klebsiella pneumoniae, confers high-level resistance to recent cephalosporins, in particular ceftazidime, and to the monobactam aztreonam. Using oligonucleotide probes, we found that the plasmid gene blaT-9 encoding TEM-9 differs from characterized blaT genes by a new combination of already known mutations. Gene blaT-9 was further studied by direct sequencing of an amplified 1.1-kb DNA fragment which contained the open reading frame and its promoter. Analysis of the nucleotide and of the deduced amino acid sequence confirmed the hybridization results and indicated that TEM-9 differs from TEM-1 by four amino acid substitutions: Phe at position 19 and Met at position 261, which have been found in TEM-4 and are known not to expand the enzyme substrate range; Lys 102, detected in TEM-3 and TEM-4, and Ser 162, present in TEM-5 and TEM-7. Each of the latter substitutions enlarges the substrate spectrum of the enzymes and they are found associated for the first time in TEM-9.