Crossed immunoelectrophoresis (CIE) has been used to characterize the soluble antigens of Mycobacterium lepraemurium (Douglas strain) (MLM). The antigen was prepared by sonification of intact bacilli, purified as described. A pool of immunoglobulin isolated from hyperimmune rabbit antisera was used as antibody. By this procedure 42 antigens from MLM could be defined in a highly reproducible manner. An analysis of crossed line immunoelectrophoresis (CLIE) and CIE with intermediate gel as methods for studying antigenic cross-reactivity are presented. Based on this analysis both BCG antigen and BCG antibody were added separately to the intermediate gel of the MLM reference system to study the cross-reactivity between this mycobacterium and MLM. It was concluded that 23 out of the 42 MLM antigens defined in our reference system showed various degrees of cross-reactivity. However, most of these cross-reactions were weak. The method appears to be well suited for studying cross-reactions between mycobacteria, but similar studies using other mycobacteria are needed before it can be decided how closely MLM is related to BCG.