Overexpression of Pseudomonas aeruginosa outer membrane protein OprH led to an 8- to 32-fold increase in susceptibility to chloramphenicol and the quinolones nalidixic acid, norfloxacin, ciprofloxacin, and fleroxacin in comparison with the susceptibility of the wild-type strain H103 grown on Mg(2+)-sufficient medium. This was true regardless of whether OprH overexpression was induced by growth of strain H103 on Mg(2+)-deficient medium, the addition of 5 mM m-toluate to cells containing the cloned oprH gene behind the inducible tol promoter in plasmid pGB25, or mutation in the polymyxin-resistant derivative strain H181. In contrast, OprH overexpression failed to reverse the quinolone resistance phenotype of a nalB mutant. OprH was purified to homogeneity by selective detergent solubilization and fast protein liquid chromatography. The addition of OprH to the solution bathing a black lipid bilayer membrane failed to give rise to an increase in membrane conductance. This suggests that OprH is not a porin but, instead, may cause increased uptake of quinolones and chloramphenicol via a non-porin pathway.