We previously demonstrated that amilorides bind to the quinone binding pocket of bovine mitochondrial complex I, not to the hitherto suspected Na⁺/H⁺ antiporter-like subunits (ND2, ND4, and ND5) [Murai, M., et al. (2015) Biochemistry 54, 2739-2746]. To characterize the binding position of amilorides within the pocket in more detail, we conducted specific chemical labeling [alkynylation (-C≡CH)] of complex I via ligand-directed tosyl (LDT) chemistry using a newly synthesized amide-type amiloride AAT as a LDT chemistry reagent. The inhibitory potency of AAT, in terms of its IC50 value, was markedly higher (∼1000-fold) than that of prototypical guanidine-type amilorides such as commercially available EIPA and benzamil. Detailed proteomic analyses in combination with click chemistry revealed that the chemical labeling occurred at Asp160 of the 49 kDa subunit (49 kDa Asp160). This labeling was significantly suppressed in the presence of an excess amount of other amilorides or ordinary inhibitors such as quinazoline and acetogenin. Taking into consideration the fact that 49 kDa Asp160 was also specifically labeled by LDT chemistry reagents derived from acetogenin [Masuya, T., et al. (2014) Biochemistry 53, 2307-2317, 7816-7823], we found this aspartic acid to elicit very strong nucleophilicity in the local protein environment. The structural features of the quinone binding pocket in bovine complex I are discussed on the basis of this finding.