For characterization of the MexCD‐OprJ efflux system of Pseudomonas aeruginosa involved in resistance to fluoroquinolones and the fourth‐generation cephems, we constructed mexC, mexD or oprJ mutants from the nfxB‐type PAO strains by insertion mutagenesis. The gene products in the resultant mutants were examined by immunoblot assay using murine and rabbit antibodies developed against purified protein and synthetic oligopeptides. Susceptibility of the mexC (MexC− MexD− OprJ−) and mexD (MexC+ MexD− OprJ−) mutants to fluoroquinolone and the fourth‐generation cephems was comparable to that of the wild‐type strain PAO1. However, the oprJ mutant (MexC+ MexD+ OprJ−) was still less susceptible than PAO1, since a MexCD‐OprM chimera system, which generated from a functional assist of the constitutively expressed OprM, can function in the efflux of the antimicrobial agents in the oprJ mutant. In fact, transformation of the oprJ mutant with an OprM‐expression plasmid decreased the former's susceptibility to the levels exhibited by the nfxB mutant without affecting the substrate specificity of MexCD‐OprJ.