A clinical isolate of Klebsiella pneumoniae was found to be resistant to ampicillin (MIC of 128 µg/ml), ticarcillin (MIC of 512 µg/ml), and ceftazidime (MIC of 128 µg/ml) and susceptible to all other -lactams; a synergistic effect between clavulanate and ceftazidime suggested the presence of an extended-spectrum -lactamase (ESBL). Transconjugants in Escherichia coli were obtained at low levels (10⁷ per donor cell) and exhibited a similar -lactam resistance pattern (resistant to ampicillin, ticarcillin, and ceftazidime at 64 µg/ml). The ESBL, pI 7.6, was encoded by a large plasmid (>100 kb) which did not carry any other resistance determinant. The ESBL-encoding gene was amplified by PCR using bla_SHV-specific primers and was sequenced. The deduced amino acid sequence of the SHV-16 ESBL showed that it differed from SHV-1 by only a pentapeptide insertion (163DRWET167) corresponding to a tandem duplication in the omega loop. The implication of the 163a-DRWET163b-DRWET sequence in ceftazidime resistance was confirmed by cloning either bla_SHV-1 or bla_SHV-16 in the same vector, subsequently introduced in the same E. coli strain. Under these isogenic conditions, SHV-16 conferred a 32-fold increase in ceftazidime MIC compared to that with SHV-1. Furthermore, site-directed mutagenesis experiments modifying either E166aA or E166bA revealed that the functional glutamic residue was that located in the first copy of the duplicated sequence. But surprisingly, the second E166b also conferred a low-level resistance to ceftazidime. This work is the first description of a class A enzyme exhibiting an extended substrate specificity due to an insertion instead of a nucleotide substitution(s) in a clinical isolate.