Purified flagella from multiple isolates of Bacillus piliformis were obtained and examined by electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (immunoblot) analyses were used to assess the purity, antigenicity, and cross-reactivity of purified flagellar preparations. SDS-PAGE demonstrated a single, major protein band evident at approximately 53 to 56 kDa in all isolates tested. Results of Western blot analyses indicated a lack of cross-reactivity between flagellar antigens and heterologous isolates. Enzyme-linked immunosorbent assays (ELISAs) were used to compare the efficacies of flagellar preparations from the various isolates as antigens in detecting B. piliformis serum antibodies from several host species. ELISA results indicated that no single flagellar preparation could be relied on to consistently identify serum antibodies in all the host species tested; however, ELISAs that utilized a trivalent flagellar antigen preparation were shown to be specific and sensitive for the detection of antibodies to B. piliformis.