The capacity of Neisseria gonorrhoeae to resist structurally diverse hydrophobic agents (HAs) because of the mtr (multiple transferable resistance) efflux system was found to be regulated at the level of transcription by two distinct mechanisms. This was surmised because a deletion that removed > 90% of the coding sequence of the mtrR (multiple transferrable resistance regulator) gene or a single-base-pair deletion within a 13-bp inverted repeat sequence located in its promoter resulted in altered expression of the mtrC gene; mtrC encodes a 44-kDa membrane lipoprotein essential for the efflux of HAs. However, the single-base-pair deletion had the more significant impact on gene expression since it resulted in the loss of expression of mtrR and a threefold increase in the expression of mtrC. Hence, the mtr efflux system in gonococci is subject to both MtrR-dependent and MtrR-independent regulation, and the levels of mtrC mRNA correlate well with HA resistance levels in gonococci.