The mtr (multiple transferable resistance) gene complex in Neisseria gonorrhoeae encodes an energy-dependent efflux pump composed of the MtrC-MtrD-MtrE cell envelope proteins that serves to export structurally diverse antimicrobial, hydrophobic agents (HAs). Many of these agents have membrane-acting detergent activity. Using Triton X-100 (TX-100) as a representative HA, we found that the mtrCDE efflux pump operon could be induced to higher levels of expression when an HA-sensitive strain was exposed to sublethal concentrations of this non-ionic detergent and the structurally related spermicide, nonoxynol-9. This induction was at the level of mtrCDE gene transcription and was independent of the MtrR repressor, which normally decreases mtrCDE gene expression. However, the enhanced resistance of gonococci to TX-100 was dependent on the expression of a previously undescribed gonococcal protein that belonged to the AraC/XylS family of transcriptional activators. We have termed this protein MtrA to signify its likely role in the activation of mtrCDE gene expression. Taken together with previous studies dealing with the genetic control of mtrCDE gene expression, we propose that gonococci can modulate their resistance to HAs through both positive and negative transcriptional control processes. The action of these regulatory processes is probably of importance in determining the survival capacity of gonococci at mucosal surfaces that contain detergent-like HAs.