When Mycobacterium tuberculosis was grown on Sauton medium, intracellular tuberculin-active protein was produced. This product was purified by chromatography on DEAE-cellulose and Sephadex G-200 and was obtained in crystalline form. The crystals were plates, somewhat irregular in shape, about 50 mum in length and 25 mum in width. The yield corresponded to a 1.5% over-all recovery of total protein. Ultracentrifugal analysis showed only one major component with a calculated molecular weight of 9700. Sedimentation velocity analysis gave a sedimentation coefficient at 20 degrees (see article) of 1.73 S. The estimated specific activities of tuberculin-active protein were 6.33 times 10-9 tuberculin units per mg of protein-nitrogen for sensitized guinea pigs and 6.33 times 10-11 tuberculin units per mg of protein-nitrogen for humans. This is the most potent tuberculin-active protein that has yet been obtained.