A highly purified protein, named MPB70, was isolated from the culture filtrate of Mycobacterium bovis BCG. This protein accounted for more than 10% of the proteins secreted into the culture medium. MPB70 was purified by precipitation with ammonium sulfate, followed by treatment with diethylaminoethyl ion exchanger, with or without 3 M urea, and by gel filtration. The final MPB70 preparation was homogenous as judged by several analyses. The molecular weight was estimated to be 18,000 by electrophoresis or molecular sieve and 15,100 by sedimentation equilibrium. The preparation did not contain sugars. The amino acid composition did not include cysteine or tryptophan. In skin reaction, MPB70 was a strictly BCG-specific antigen and, among the guinea pigs sensitized with the heat-killed cells of the various species of mycobacteria--Mycobacterium tuberculosis strains H37Rv and Aoyama B, Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium phlei, and BCG, it elicited a delayed cutaneous reaction only in the guinea pigs sensitized with BCG. The potency of MPB70 in the skin reaction was about one-twentieth of the standard purified protein derivative.