A reference system for the soluble antigens of Mycobacterium bovis, strain BCG is described. The system is based on crossed immunoelectrophoresis with intermediate gel. A commercially available immunoglobulin preparation made from rabbit anti-BCG hyperimmune serum was used as reference antibodies, while a concentrated BCG culture filtrate was used as reference antigen. The pattern obtained was highly reproducible, and most of the components were stable when the fiftyfold-concentrated culture filtrate was stored at -20 degrees C. About thirty different antigenic components were selected as reference antigens and numbered. The majority of the reference antigens were present in extracts prepared from BCG by ultrasonication or bacterial press extraction. Use of the system for studies of antigenic relationship between mycobacteria, identification and quantification of antigens, and characterization of antimycobacterial antibodies are illustrated by examples. The antigens of two preparations of tuberculin purified protein derivative (PPD) were identified. The antigen designated BCG60 was found to be a najor constituent of tuberculin PPD. Evidence is presented that this antigen is cell wall associated.