Numerous hydrophobic and amphipathic compounds including several detergents are known to inhibit the ubiquinone reductase reaction of respiratory chain complex I (proton pumping NADH:ubiquinone oxidoreductase). Guided by the X-ray structure of the peripheral arm of complex I from Thermus thermophilus we have generated a large collection of site-directed mutants in the yeast Yarrowia lipolytica targeting the proposed ubiquinone and inhibitor binding pocket of this huge multiprotein complex at the interface of the 49-kDa and PSST subunits. We could identify a number of residues where mutations changed I(50) values for representatives from all three groups of hydrophobic inhibitors. Many mutations around the domain of the 49-kDa subunit that is homologous to the [NiFe] centre binding region of hydrogenase conferred resistance to DQA (class I/type A) and rotenone (class II/type B) indicating a wider overlap of the binding sites for these two types of inhibitors. In contrast, a region near iron-sulfur cluster N2, where the binding of the n-alkyl-polyoxyethylene-ether detergent C(12)E(8) (type C) was exclusively affected, appeared comparably well separated. Taken together, our data provide structure-based support for the presence of distinct but overlapping binding sites for hydrophobic inhibitors possibly extending into the ubiquinone reduction site of mitochondrial complex I.