Dicyclohexylcarbodiimide (DCCD), an inhibitor of proton translocation, has been shown to bind preferentially to the myelin proteolipid and to inhibit proton movement in liposomes containing the proteolipid (Lin, L. and Lees, M. 1982. Proc. Natl. Acad. Sci. USA 79:941-945). In the present study the location of the DCCD-binding site(s) in the sequence of the myelin proteolipid has been investigated. Of the 11 dicarboxylic acid residues theoretically available for binding, Asp 149 has been positively identified as a binding site. Seven dicarboxylic acid residues have essentially been ruled out as binding sites and one site has been tentatively ruled out. The status of the two remaining sites has not been determined.