Exchangeable apolipoproteins are proteins that reversibly bind lipoprotein particles to facilitate their transport in vivo. The structure/function relationship of apolipophorin-III (apo-III), the only insect exchangeable apolipoprotein, has been investigated by examining the association of this protein with lipid vesicles. The importance of a conserved leucine residue, reported to be essential for apo-III binding to lipids, has been evaluated through site-directed mutagenesis. A unique cysteine replaces the conserved leucine at position 30 in recombinant apo-III (L30C protein). This substitution results in the covalent dimerization of the apo-III mutant via a disulfide bond. The cysteine mutation causes no difference in surface hydrophobicity of the L30C proteins when compared to the wild type apo-III. Wild type apo-III, L30C monomer, and L30C dimer associate with dimyristoylphosphatidylcholine (DMAC) vesicles in a similar manner, resulting in a reduction of turbidity of a phospholipid vesicle suspension. Analysis with transmission electron microscopy (TEM) reveals disk-like complexes identical to those previously reported with the wild type apo-III. Because the mutation of the conserved leucine seems to affect the solution behavior and surface hydrophobicity of apo-III, this residue is likely to be exposed to the aqueous environment. However, the similar behaviors of the wild type protein, the L30C monomer, and L30C dimer with respect to the binding of phospholipid vesicles suggest that this residue is not absolutely required for the protein binding to hydrophobic or amphiphilic interfaces.