BACKGROUND: The efficacy of benzimidazole fungicides is often limited by resistance, and this is the case with the use of carbendazim for controlling Fusarium head blight caused by Gibberella zeae (Schwein.) Petch (anamorph Fusarium graminearum). Recent studies have shown that carbendazim resistance in field strains of G. zeae is associated with mutations in the β(2)-tubulin gene. The aims of the present study were to validate this mechanism and research the binding sites of carbendazim on β(2)-tubulin. RESULTS: This work used site-directed mutagenesis followed by gene replacement to change the β(2)-tubulin gene of a carbendazim-sensitive field strain of G. zeae at residues 50, 167, 198 or 200. The transformants were confirmed and tested for their sensitivity to carbendazim. All the mutants were resistant to carbendazim, but the level of resistance differed depending on the mutation. Biological characteristics did not differ between the field strain and the site-directed mutants. A three-dimensional model of β(2)-tubulin was constructed, and the possible carbendazim binding site was analysed. CONCLUSION: Mutations at codons 50, 167, 198 and 200 of G. zeae β(2)tub could cause resistance to carbendazim, and these codons may form a binding pocket.