A B S T R A C T The noncytotoxic immunosuppressive substance detected in crude extracellular products of Streptococcus intermedius (CEP-Si) was fractionated by two steps of preparative isoelectric focusing in sucrose gradients using ampholytes of pH range from 3.5 to 6 and 4 to 5, respectively. The in vitro and in vivo suppressor effects of the most highly purified fraction of CEP-Si, designated fraction 3' (F3'EP-Si), corresponded well with those of the original CEP-Si. F3'EP-Si was sensitive to the effects of alpha, gammia, and delta chymotrypsin, trypsin, and heating. It contained -1% of the total amount of protein found in the original CEP-Si, corresponding to a single band on analytical isoelectric focusing, stainable by Coomassie Blue and of isoelectric point of 4.25. The absorption spectrum of F3'EP-Si had a maximum at 260 nm but its biological activity was resistant to deoxyribonuclease and ribonuclease A and it did not contain mlaterial stainable by methylene blue. It was also resistant to neuraminidase and did not contain material stainable by periodic acid Schiff. We conclude that the substance responsible for the suppressor activity of CEPSi is a protein of molecular weight -90,000, which adheres to Sephadex or cellulose acetate and forms complexes with other, nonactive constituents of