In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCDI-3 possesses the NH2-terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that several eukaryotes have ABCD protein homologs lacking the NH2-terminal hydrophobic segment (HO motif). To investigate whether the role of the NH2-terminal HO motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH2-terminal HO motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH2-terminal HO motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH2-terminal HO motif in organelle targeting is widely conserved in living organisms. (C) 2014 Elsevier Inc. All rights reserved.