Flavobacterium johnsoniae CIP100931 is resistant to most beta-lactam antibiotics and has a decreased susceptibility to carbapenems. A beta-lactamase gene was cloned and expressed in Escherichia coli DH10B. The purified beta-lactamase, JOHN-1, with a pI value of 9.0 and with a determined relative molecular mass of approximately 27 kDa was found to be a monomeric zinc-dependent enzyme that hydrolyses penicillins, narrow- and expanded-spectrum cephalosporins, carbapenems, but not monobactams. Sequence analysis revealed that JOHN-1 is a molecular class B beta-lactamase that is most closely related to BlaB from Chryseobacterium meningosepticum and IND-1 from Chryseobacterium indologenes (47% and 41% amino acid identity, respectively). JOHN-1 is a new member of the highly divergent subclass B1 lineage of metallo-enzymes. Although F. johnsoniae and Chryseobacterium spp. are phylogenetically related bacteria, this report further underlines the heterogeneity of class B beta-lactamases that are naturally produced by environmental Gram-negative aerobes and that are now recognized as the most important reservoir for these beta-lactamase genes.