The mtr (multiple transferable resistance) system of Neisseria gonorrhoeae mediates resistance of gonococci to structurally diverse hydrophobic agents (HAs) through an energy-dependent efflux process. Recently, complete or partial ORFs that encode membrane proteins (MtrC, MtrD, MtrE) forming an efflux pump responsible for removal of HAs from gonococci were identified and appeared to constitute a single transcriptional unit. In this study, the complete nucleotide sequence of the mtrD gene was determined, permitting the characterization of the MtrD protein. The full-length MtrD protein has a predicted molecular mass of nearly 114 kDa, putatively containing a 56 amino acid signal peptide. MtrD displays significant amino acid sequence similarity to a family of cytoplasmic membrane proteins, termed resistance/nodulation/division (RND) proteins, which function as energy-dependent transporters of antibacterial agents and secrete bacterial products to the extracellular fluid. The predicted topology of the MtrD transporter protein revealed 12 potential membrane-spanning domains, which were clustered within the central and C-terminal regions of the primary sequence. Loss of MtrD due to insertional inactivation of the mtrD gene rendered gonococci hypersusceptible to several structurally diverse HAs, including two fatty acids (capric acid and palmitic acid) and a bile salt (cholic acid), but not hydrophilic antibiotics such as ciprofloxacin and streptomycin. Since gonococci often infect mucosal sites rich in toxic fatty acids and bile salts, the expression of the mtr efflux system may promote growth of gonococci under hostile conditions encountered in vivo.