Abstract: A positive plasmid that comprises the entire floR ORF and possible regulatory site was obtained and named plasmid pGEM-floR. The recombinant plasmids were successfully transformed into E. coli JM109. A significant resistance to florfenicol and chloramphenicol was found in this strain which was susceptive to tetracycline and gentamicin. Location of FloR protein was confirmed by immunoblotting that using the membrane fraction of florfenicol-resistant E. coli strains (pGEMfloR/JM109 and CVM1841) and the florfenicol-sensitive (negative-control) strains (pGEM-T/JM109) with the GST-FloR1 mouse antisera. The Western blot results showed that the antibody had specific binding to the FloR protein and confirmed that FloR protein was a membrane protein in florfenicol-resistant bacteria. The research shows that protein encoded by floR gene locates at the cellular membrane and confers crossresistant to flofenicol and chloramphenicol.