Protein and antigenic heterogeneity among isolates of Bacillus piliformis, the etiologic agent of Tyzzer's disease, were investigated. The seven isolates utilized in this study were originally isolated from naturally infected animals of different animal species and diverse geographical locations. Isolates were propagated in mammalian cell lines, and bacterial extracts were prepared. Protein and antigenic profiles were compared among isolates, using Coomassie blue-stained polyacrylamide gels and Western blot (immunoblot) analyses, respectively. Results showed differences in protein and antigen banding patterns, indicating diversity among isolates. Western blots probed with serum preabsorbed with a heterologous bacterial extract revealed that numerous antigens have different electrophoretic mobilities among isolates but apparently share common epitopes. Immunodominant cross-reactive antigens may be candidate proteins useful for development of improved serologic diagnostic tests, allowing identification of animals infected with a wide range of B. piliformis isolates.