The purpose of this study was to localize antigenic regions on the flagellin protein of Campylobacter jejuni 81116. This strain has two flagellin genes, flaA and flaB, which are 95% identical; only flaA seems to be expressed in motile C. jejuni 81116 cells. Fragments of flaA and flaB were cloned in the bacterial expression vector pEX, and the expression products were incubated with flagellin-specific antibodies. Monoclonal antibodies to broadly cross-reactive epitopes recognized fragments that are located in the termini (CF16 and CF17) and in the center (CF15) of both flagellin A and B proteins. Most of the serotype-specific monoclonal antibodies (CF1, CF2, CD3, CF4, and CF13) reacted with only the center of flagellin A in an area where flagellin A and B differ in 6 amino acid residues. The epitopes in this area were further characterized by competitive binding experiments. The charge and molecular weight microheterogeneity of flagellin, as determined by two-dimensional gel electrophoresis, were unrelated to the expression of both flagellin genes or parts thereof.