Genes encoding carbapenemases in 15 reference strains of Chryseobacterium (Flavobacterium) meningosepticum from the United Kingdom National Collection of Type Cultures and in one recent clinical isolate were investigated. All the strains hydrolyzed imipenem, but their levels of resistance to carbapenems varied, with imipenem and meropenem MICs ranging from 2 to >32 microg/ml. The blaB gene, which encodes a molecular-class B carbapenemase, was detected in only six reference strains and in clinical isolate 97/P/5448. The gene from 97/P/5448 had 98% nucleotide identity with the published sequence of blaB (from strain NCTC 10585) and was designated blaB2. A distinct carbapenemase gene, designated blaB3, was cloned from the type strain of C. meningosepticum, NCTC 10016. blaB3 had an open reading frame of 750 bp with 82% nucleotide identity to blaB and blaB2 and encoded a beta-lactamase of 249 amino acids, including the putative signal peptide. This beta-lactamase showed 87.6 and 86.7% amino acid homology with BlaB and BlaB2, respectively. blaB3 was detected in one other reference strain besides NCTC 10016, but the genetic basis of the carbapenemase activity detected in the other seven reference strains was not defined. Thus, neither blaB nor blaB3 was ubiquitous in the strains of C. meningosepticum studied, indicating that the reference strains may represent more than one bacterial species, each with its own intrinsic metallo-beta-lactamase. Further taxonomic studies of C. meningosepticum are necessary to resolve this topic. Chryseobacterium spp. are environmental organisms and occasional opportunist pathogens. They apparently represent a reservoir of diverse metallo-beta-lactamases, which potentially spread to gram-negative bacteria of greater clinical significance.