<jats:p>The effect of ten naturally occurring and two synthetic inhibitors of NADH:ubiquinone oxidoreductase (complex I) of bovine heart, <jats:italic>Neurospora crassa</jats:italic> and <jats:italic>Escherichia coli</jats:italic> and glucose:ubiquinone oxidoreductase (glucose dehydrogenase) of <jats:italic>Gluconobacter oxidans</jats:italic> was investigated. These inhibitors could be divided into two classes with regard to their specifity and mode of action. Class I inhibitors, including the naturally occuring piericidin A, annonin VI, phenalamid A<jats:sub>2</jats:sub>, aurachins A and B, thiangazole and the synthetic fenpyroximate, inhibit complex I from all three species in a partially competitive manner and glucose dehydrogenase in a competitive manner, both with regard to ubiquinone. Class II inhibitors including the naturally occuring rotenone, phenoxan, aureothin and the synthetic benzimidazole inhibit complex I from all species in an non‐competitive manner, but have no effect on the glucose dehydrogenase. Myxalamid PI could not be classified as above because it inhibits only the mitochondrial complex I and in a competitive manner. All inhibitors affect the electron‐transfer step from the high‐potential iron‐sulphur cluster to ubiquinone. Class I inhibitors appear to act directly at the ubiquinone‐catalytic site which is related in complex I and glucose dehydrogenase.</jats:p>