In man the early immune response in Lyme disease is primarily directed against the endoflagellin antigen. Isolated flagellar protein of Borrelia burgdorferi suggests itself as a suitable test antigen. However, cross-reactivity between flagellins of B. burgdorferi, Escherichia coli, Bacillus subtilis, Proteus mirabilis and Salmonella typhimurium was demonstrated by immunoblotting and ELISA with polyclonal rabbit-hyperimmune-sera. Tryptic cleavage of recombinant B. burgdorferi 41 kDa flagellin, expressed in E. coli, produced a peptide fragment which was recognized exclusively by antisera to Borrelia species. This peptide was designated as the 14 kDa fragment due to its migratory behaviour in SDS-PAGE. The fragment is part of the variable region of the flagellin, as proven by amino acid sequencing. The flagellin peptide was employed as an antigen in ELISA and immunoblot assays, testing the polyclonal sera mentioned above. The specificity was superior to that obtained with the intact recombinant flagellin.