Low-level resistance of Neisseria gonorrhoeae to toxic hydrophobic agents (HAs), including some antibiotics, is chromosomally mediated via the multiple transferable resistance (mtr) efflux system. The gene encoding the 48:3 kDa outer-membrane protein MtrE, which is associated with the mtr phenotype, was identified and is homologous to export-associated outer-membrane proteins, including the OprM (formerly OprK) lipoprotein of Pseudomonas aeruginosa. Insertional inactivation of the mtrE gene in N. gonorrhoeae strain FA19 resulted in the loss o the outer-membrane protein, with concomitant hypersusceptibility of the mutant strain to a range of HAs. The properties of this mutant confirmed the role of MtrE in multidrug resistance mediated by an active efflux mechanism. Secondary structure predictions for MtrE indicated a largely hydrophilic protein with a single alpha-helical transmembrane region. A transposon-like element, similar to that found downstream of the region containing the promoters for mtrR and mtrC in Neisseria meningitidis, was identified 63 bp downstream of the mtrE gene.