Spontaneous cefsulodin-resistant mutants of Pseudomonas aeruginosa PAO4089 were isolated on agar impregnated with 3 mg/l of cefsulodin. This strain does not produce any chromosomal beta-lactamase. The MICs of cefsulodin for the parent and its mutants were 0.78 and 12.5 mg/l, respectively. Complete cross-resistance between cefsulodin and seven other antipseudomonal beta-lactams was noted in the mutants. The mutant gene, designated as pbpB, was mapped by FP5 plasmid-mediated conjugation and found to be near to cys-59 on the PAO chromosome, the gene order being pur-67, oruI, pbpB and cys-59. There were no detectable differences between the parent and its mutants in their outer membrane protein profiles. Penicillin-binding protein assay, by the competition method, with cefsulodin or carbenicillin showed a significant reduction in affinity of PBP3 for these beta-lactams. This PBP is the primary target for cefsulodin in P. aeruginosa. The genetic mechanism by which the cefsulodin-resistant clinical isolates of P. aeruginosa have emerged is discussed.