ABSTRACT In the course of surveying for the carbapenem-hydrolyzing metallo-β-lactamase gene bla _IMP in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo-β-lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the gene bla _IMP-3 was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of bla _IMP-3 was identical to that of bla _IMP-1 , except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid bla genes were constructed from bla _IMP-3 and bla _IMP-1 by recombinant DNA techniques, and β-lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of the bla _IMP-3 gene. This indicated that extension of the substrate profile in the metallo-β-lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene bla _IMP-3 is an ancestor of bla _IMP-1 .