The mtr (multiple transferable resistance) system of Neisseria gonorrhoeae determines levels of gonococcal resistance to hydrophobic agents (HAs), including detergent-like fatty acids and bile salts that bathe certain mucosal surfaces. The genetic organization of the mtr system was determined and found to consist of the mtrR gene, which encodes a transcriptional regulator (MtrR), and three tandemly linked genes termed mtrCDE. The mtrCDE genes were organized in the same apparent transcriptional unit, upstream and divergent from the mtrR gene. The mtrCDE-encoded proteins of N. gonorrhoeae were analogous to a family of bacterial efflux/transport proteins, notably the MexABOprK proteins of Pseudomonas aeruginosa and the AcrAE and EnvCD proteins of Escherichia coli, that mediate resistance to drugs, dyes, and detergents. Inactivation of the mtrC gene resulted in loss of the MtrC lipoprotein and rendered gonococci hypersusceptible to structurally diverse HAs; this revealed the importance of the mtr system in determining HAR in gonococci. Further support for a role of the mtrCDE gene complex in determining levels of HAR in gonococci was evident when transformants bearing mutations in the mtrR gene were analysed. In this respect, missense and null mutations in the mtrR gene were found to result in increased levels of MtrC and HAR. However, high levels of MtrC and HAR, similar to those observed for clinical isolates, were associated with a single bp deletion in a 13 bp inverted repeat sequence that intervened the divergent mtrR and mtrC genes. We propose that the 13 bp inverted-repeat sequence represents a transcriptional control element that regulates expression of the mtrRCDE gene complex, thereby modulating levels of gonococcal susceptibility to HAs.