A defined medium was developed in which easily measured quantities of exotoxin A (PE) were produced by Pseudomonas aeruginosa PA-103. The medium contained three L-amino acids (arginine, aspartic acid, and alanine), basal and trace salts including 14 mM K2HPO4, 14 mM glucose, and 140 mM glycerol. The concentrations of amino acids which yielded most satisfactory results were 6 mM alanine, 13 mM aspartic acid, and 16 mM arginine. The identity of PE in the culture supernatant fluid was demonstrated by adenosine diphosphate-ribosyl transferase activity and by immunodiffusion with sheep antitoxin elicited with purified PE and with PE produced in Trypticase soy broth dialysate and pure PE as controls. PE production was also demonstrated by mouse lethality and passive hemagglutination. As compared to Trypticase soy broth dialysate, P. aeruginosa produced 25 to 50% PE in the defined medium. Different strains of P. aeruginosa produced PE in the defined medium in proportions relative to those in Trypticase soy broth dialysate.