Sera from rabbits immunized with sonicates of Mycobacterium bovis BCG were passed through an immunoadsorbent made of a soluble BCG extract to make partially purified antibodies to BCG. These antibodies were in turn used to prepare an immunoadsorbent through which the BCG extract was passed. The partially purified antigenic material was radiolabeled and subjected to electrophoresis in acrylamide gels. One of the radiolabeled fractions isolated (BCG-C) was found to bind to antibodies to BCG and H37Rv, but not to antibodies in sera from rabbits immunized with other mycobacterial species or Nocardia asteroides. The reaction between BCG-C and the partially purified antibodies to BCG was inhibited by small amounts of different BCG antigens. Cultures obtained from 25 patients with tuberculous diseases, other bacterial cultures, and various bacterial extracts were tested for their capacity to inhibit this reaction. Each of 13 mycobacteria identified as M. tuberculosis inhibited this reaction. Equivalent numbers of 12 strains of mycobacteria other than M. tuberculosis and high concentrations of other bacterial extracts did not inhibit, indicating that determinants of BCG present in M. tuberculosis were not detected in the other mycobacteria or in non-acid-fast bacteria. The use of sequential purification procedures could be of potential clinical value in quickly differentiating between M. tuberculosis and a variety of other mycobacteria.