In order to provide a better system for investigating the role of cytotoxin in pathogenesis, we mutated wild-type Pseudomonas aeruginosa PA158 by introducing a transposon. The resulting pool of mutants was screened for cytotoxin-deficient strains. One mutant strain, PA114F5, was compared with PA158. Except for cytotoxin production and antibiotic resistance (specified by the transposon), the two strains appear isogenic. This mutant strain should be useful in further clarifying the role of cytotoxin in pathogenesis.