A putative multidrug efflux pump, EmrD-3, belonging to the major facilitator superfamily (MFS) of transporters and sharing homology with the Bcr/CflA subfamily, was identified in Vibrio cholerae O395. We cloned the emrD-3 gene and evaluated its role in antimicrobial efflux in a hypersensitive Escherichia coli strain. The efflux activity of this membrane protein resulted in lowering the intracellular concentration of ethidium. The recombinant plasmid carrying emrD-3 conferred enhanced resistance to several antimicrobials. Among the antimicrobials tested, the highest relative increase in minimum inhibitory concentration (MIC) of 102-fold was observed for linezolid (MIC = 256 microg/ml), followed by an 80.1-fold increase for tetraphenylphosphonium chloride (TPCL) (156.2 microg/ml), 62.5-fold for rifampin (MIC = 50 microg/ml), >30-fold for erythromycin (MIC = 50 microg/ml) and minocycline (MIC = 2 microg/ml), 20-fold for trimethoprim (MIC = 0.12 microg/ml), and 18.7-fold for chloramphenicol (MIC = 18.7 microg/ml). Among the fluorescent DNA-binding dyes, the highest relative increase in MIC of 41.7-fold was observed for ethidium bromide (125 microg/ml) followed by a 17.2-fold increase for rhodamine 6G (100 microg/ml). Thus, we demonstrate that EmrD-3 is a multidrug efflux pump of V. cholerae, the homologues of which are present in several Vibrio spp., some members of Enterobacteriaceae family, and Gram-positive Bacillus spp.