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SUMMARY: Carbenicillin resistance in two strains of Pseudomonas aeruginosa has been investigated. Pseudomonas aeruginosa NCTC 10490, a carbenicillin sensitive strain, produced a β-lactamase when grown in the presence of carbenicillin. This enzyme although active against benzylpenicillin did not hydrolyse carbenicillin. Cell walls of P. aeruginosa NCTC 10490 habituated to the presence of carbenicillin showed a higher lipid content compared with the walls of the parent strain. Hospitalisolated, carbenicillin-resistant P. aeruginosa 69/4992 synthesized a β-lactamase which hydrolysed carbenicillin as well as benzylpenicillin. The two types of enzyme were characterized by their substrate profile, sensitivity to enzyme inhibitors and resolution by isoelectric focusing. Enzymes active against carbenicillin were differentiated by isoelectric focusing from those unable to hydrolyse carbenicillin; both types of enzyme were shown to be complex mixtures by this technique.