The importance of the mtrCDE-encoded efflux pump in conferring chromosomally mediated penicillin resistance on certain strains of Neisseria gonorrhoeae was determined by using genetic derivatives of penicillin-sensitive strain FA19 bearing defined mutations (mtrR, penA, and penB) donated by a clinical isolate (FA6140) expressing high-level resistance to penicillin and antimicrobial hydrophobic agents (HAs). When introduced into strain FA19 by transformation, a single base pair deletion in the mtrR promoter sequence from strain FA6140 was sufficient to provide high-level resistance to HAs (e.g., erythromycin and Triton X-100) but only a twofold increase in resistance to penicillin. When subsequent mutations in penA and porIB were introduced from strain FA6140 into strain WV30 (FA19 mtrR) by transformation, resistance to penicillin increased incrementally up to a MIC of 1.0 micro g/ml. Insertional inactivation of the gene (mtrD) encoding the membrane transporter component of the Mtr efflux pump in these transformant strains and in strain FA6140 decreased the MIC of penicillin by 16-fold. Genetic analyses revealed that mtrR mutations, such as the single base pair deletion in its promoter, are needed for phenotypic expression of penicillin and tetracycline resistance afforded by the penB mutation. As penB represents amino acid substitutions within the third loop of the outer membrane PorIB protein that modulate entry of penicillin and tetracycline, the results presented herein suggest that PorIB and the MtrC-MtrD-MtrE efflux pump act synergistically to confer resistance to these antibiotics.