Cross-reactions between five proteins actively secreted by Mycobacterium tuberculosis were studied by crossed immunoelectrophoresis, SDS-PAGE with immunoblotting, and ELISA using polyclonal rabbit antisera and mouse monoclonal antibodies to the purified proteins. The monoclonal antibody HBT4 was demonstrated to react with the MPT51 protein. The 85A, 85B and 85C constituents of the M. tuberculosis and Mycobacterium bovis BCG antigen 85 complex cross-react extensively, each of the components containing component-specific as well as cross-reacting epitopes. These components also cross-reacted with MPT51 and MPT64. N-terminal sequence studies revealed striking homology at the amino acid level between 85A, 85B, 85C and MPT51. MPT64 showed less homology. In addition, striking homology was demonstrated between two different stretches within the 85B sequence and indicated between three stretches within the MPT64 molecule. Thus, a family of at least four secreted proteins with common structural features has been demonstrated in mycobacteria. MPT64 may also belong to this family.