1. Streptolysin O, an exotoxin produced by group A beta-hemolytic streptococci, has been purified from Streptococcus pyogenes culture supernatants. 2. The isolation and purification procedure involved ammonium sulphate and polyethylene glycol precipitations, and ion-exchange chromatographies on CM-Sepharose and Mono Q. 3. The proposed procedure introduces two ion-exchange chromatography steps making the purification process simpler and, especially, more reproducible than other described protocols. 4. The purified streptolysin O was hemolytically active, had a specific activity of 415,000 HU/mg, an optimum pH of 7.0, a relative molecular mass of 60,100 and an isoelectric pH of 7.5.