The cloned oprH gene of Pseudomonas aeruginosa was mutated by inserting a 1.4-kbp fragment that encodes tetracycline resistance. This mutated gene was then incorporated into the P. aeruginosa chromosome by homologous recombination. Growth of the resultant oprH::tet mutant in Mg(2+)-deficient medium had little effect on susceptibility to polymyxin B, gentamicin, or EDTA unless the mutant was complemented by the cloned oprH gene in plasmid pGB25. In contrast, growth of the parent strain on Mg(2+)-deficient medium resulted in resistance to all three agents. These data support the hypothesis that overexpression of OprH under Mg(2+)-deficient growth conditions is conditionally required for resistance to these three agents.