Purpose: Determination of the effect of 4, 4[prime ]-diisothiocyano-2, 2[prime ]-stilbene disulfonate (DIDS) on the mitochondrial ADP/ATP carrier (AAC).Methods: Bovine heart mitochondria and their inverted vesicles were treated with various concentrations of DIDS for 10 min at 25[deg ]C.Results: DIDS inhibited ADP uptake via AAC of inverted vesicles, but not mitochondria. Of the mitochondrial membrane proteins, DIDS was found to interact specifically only with AAC. Treatment of the vesicles with DIDS inhibited the specific labeling of Cys 1 5 9 by eosin-5-maleimide (EMA), although DIDS modified only Lys 2 2 . In addition, labeling by DIDS was completely inhibited by pretreatment of the vesicles with bongkrekic acid, which is a transport inhibitor acting from the matrix side, but not affected by treatment with carboxyatractyloside, which is a transport inhibitor acting from the cytosolic side, did not affect labeling by DIDS.Conclusions: DIDS specifically labeled Lys 2 2 located in the putative first transmembrane segment of AAC from the matrix side. As Cys 1 5 9 is located in the second loop facing the matrix space (loop M2), the inhibition by DIDS of the labeling of Cys 1 5 9 by EMA suggests that the region containing Lys 2 2 in this segment interacts with loop M2.