Human ATP-binding cassette transporter isoform B6 (ABCB6) has been proposed to be situated in both the inner and outer membranes of mitochondria. These inconsistent observations of submitochondrial localization have led to conflicting interpretation in view of directions of transport facilitated by ABCB6. We show here that ABCB6 has an N-terminal hydrophobic region of 220 residues that functions as a-primary determinant of co-translational targeting to the endoplasmic reticulum (ER), but it does not have any known features of a mitochondrial targeting sequence. We defined the potential role of this hydrophobic extension of ABCB6 by glycosylation site mapping experiments, and demonstrated that the first hydrophobic segment acts as a type I signal-anchor sequence, which mediates N-terminal translocation through the ER membrane. Laser scanning microscopic observation revealed that ABCB6 did not co-localize with mitochondrial staining. Rather, it localized in the ER-derived and brefeldin A-sensitive perinuclear compartments, mainly in the Golgi apparatus. (c) 2008 Elsevier Inc. All rights reserved.