BACKGROUND: Ametoctradin is an agricultural fungicide that inhibits the mitochondrial bc₁ complex of oomycetes. The bc₁ complex has two quinone binding sites that can be addressed by inhibitors. Depending on their binding sites and binding modes, the inhibitors show different degrees of cross‐resistance that need to be considered when designing spray programmes for agricultural fungicides. The binding site of ametoctradin was unknown. RESULTS: Cross‐resistance analyses, the reduction of isolated Pythium sp. bc₁ complex in the presence of different inhibitors and molecular modelling studies were used to analyse the binding site and binding mode of ametoctradin. All three approaches provide data supporting the argument that ametoctradin binds to the Pythium bc₁ complex similarly to stigmatellin. CONCLUSION: The binding mode of ametoctradin differs from other agricultural fungicides such as cyazofamid and the strobilurins. This explains the lack of cross‐resistance with strobilurins and related inhibitors, where resistance is mainly caused by G143A amino acid exchange. Accordingly, mixtures or alternating applications of these fungicides and ametoctradin can help to minimise the risk of the emergence of new resistant isolates. © 2015 Society of Chemical Industry