MPB70 is a constituent of Mycobacterium bovis BCG, which accounts for about 10% of the total protein content in the nutrient fluid after culture on Sauton medium. Previous studies of cell-mediated immune reactions have shown that MPB70 is a highly BCG-specific antigen occurring in markedly different concentrations in different substrains of BCG. The present studies were based on the reactivity of MPB70 with antibodies. The precipitate caused by MPB70 was identified in the crossed immunoelectrophoresis pattern of sonicates and concentrated culture fluids of BCG Tokyo. The antigen was quantified by radioimmunoassay inhibition tests on culture fluids standardized on the basis of identical total protein content. It was present in high concentrations in BCG substrains Tokyo, Moreau, Russia, and Sweden, and in M. bovis strain Ravenel. It was also detected in BCG substrains Glaxo, Copenhagen, Tice, and Pasteur, and in M. tuberculosis H37Rv, but in the latter 4 in only 1% or less of the concentration in BCG Tokyo. Six different species of slow- and rapid-growing mycobacteria showed no evidence of cross-reactivity, but a cross-reacting antigen was demonstrated in Nocardia asteroides. The antibody response after immunization of rabbits with BCG Tokyo and BCG Copenhagen was very similar in a polyvalent assay for antibodies reacting with various components of BCG. By contrast, there was a striking difference in anti-MPB70 antibodies, which were rapidly formed in large amounts after immunization with BCG Tokyo and only in very small amounts after immunization with BCG Copenhagen.