Organomercury lyase (MerB) is a key enzyme in bacterial detoxification and bioremediation of organomercurials. However, the merB gene is often considered as an ancillary component of the mer operon because there is zero to three merB genes in different mer operons identified so far. In this study, organomercurials' removal abilities of native mercury-resistant bacteria that have one or multiple merB genes were examined. Each heterogeneous merB genes from these bacteria was further cloned into Escherichia coil to investigate the substrate specificity of each MerB enzyme. The merB1 gene from Bacillus megaterium MB1 conferred the highest volatilization ability to methylmercury chloride, ethylmercury chloride, thimerosal and p-chloromercuribenzoate, while the merB3 from B. megaterium MB1 conferred the fastest mercury volatilization activity to p-chloromercuribenzoate. The substrate specificities among these MerB enzymes show the necessity for selecting the appropriate bacteria strains or MerB enzymes to apply them in bioremediation engineering for cleaning up specific organomercurial contaminations. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.