Nitrosoguanidine mutagenesis of Pseudomonas aeruginosa strain PAO-1 yielded a mutant strain, PAO-PR1, which produced a protein that was immunologically indistinguishable from native toxin A and was nontoxic for cultured Chinese hamster ovary cells. In contrast to native toxin, the cell-associated and extracellular crossreactive material (CRM), designated "CRM protein," possessed no adenosine diphosphate-ribosylating activity. This CRM protein comigrated with native toxin A on sodium dodecyl sulfate/polyacrylamide gels, could be immunoprecipitated with antitoxin from culture supernatants of strain PAO-PR1, and gave a reaction of identity in immunological assays. Equivalent amounts of toxin A antigen and CRM protein antigen were produced in liquid culture by their respective strains as quantitated in a radioimmunoassay for toxin A. These data suggest that mutant strain PAO-PR1 possesses one or more missense mutations within the structural gene for toxin A that adversely affect enzymatic activity, thereby rendering the molecule nontoxic.