With the sequential use of dialysis, chromatography on Sephadex G-100, reactive red 120-agarose, p-hydroxymercuribenzoate-agarose, phenyl-Sepharose, poly(L-lysine)-agarose, and isoelectrofocusing, the thymocyte stimulatory factor activity of interleukin 2 from rat spleen was purified about 8,000-fold. By the same procedures, the T cell growth factor activity of interleukin 2 was purified about 6,000-fold. The major peaks of thymocyte stimulatory factor activity and T cell growth factor activity cochromatographed in the various systems used, but T cell growth factor activity without significant thymocyte stimulatory factor activity was sometimes present. Both activities were found to have a pI between pH 5.50 and 6.30. Detectable thymocyte stimulatory factor activity was found at concentrations as low as 0.1-0.2 ng of protein per 0.2 ml. Dose--response plots were linear up to at least 1 ng of protein. Preparations of interleukin 2 labeled with 125I-containing Bolton--Hunter reagent and purified by the procedure mentioned above were electrophoresed on a polyacrylamide gel under denaturing and reducing conditions. The 125I-labeled material migrated in one major band with a molecular weight under 20,000 and three smaller bands with molecular weights of about 20,000, 60,000, and 90,000. Material with thymocyte stimulatory factor activity did not bind to a number of lectin-gels.