Toxoid was prepared by treating exotoxin A of Pseudomonas aeruginosa with formalin. Coincubation of exotoxin A for two to four weeks at 37 degrees C with 0.5% formalin and 10(-3) M lysine followed by one to two weeks of storage in the absence of these reagents reduced cytotoxicity, preserved antigenicity, and minimized subsequent reversion of the toxoid. Formalin enhanced the adenosine diphosphate (ADP)-ribosyl transferase activity of the toxin while decreasing its toxicity, whereas formalin plus lysine reduced both. Although no antigenic changes were detected by immunodiffusion analysis, analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed minor alterations in the toxin structure. Toxoid induced high titers of antibody to exotoxin A in the sera of mice and rabbits. Antiserum to toxoid neutralized mouse lethality, cytotoxicity, and ADP-ribosyl transferase activity of untreated exotoxin A. Toxoid-immunized mice were resistant to large doses of exotoxin A administered iv.