70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) is a member of ATP-binding cassette (ABC) protein subfamily D. ABC subfamily D proteins are also known as peroxisomal ABC proteins. Therefore, P70R is thought to be a peroxisomal membrane protein. However, the subcellular localization of P70R is not extensively investigated. In this study, we transiently expressed P70R in fusion with HA (P70R-HA) in CHO cells and examined subcellular localization by immunofluorescence. Surprisingly, P70R-HA was localized to the endoplasmic reticulum (ER), not to peroxisomes. To examine the ER-targeting property of P70R, we expressed various NH(2)-terminal deletion constructs of P70R. Among the NH(2)-terminal deletion constructs, mutant proteins starting with hydrophobic transmembrane segment (TMS) were localized to ER, but the ones containing the NH(2)-terminal hydrophilic cytosolic domain were not. ABC subfamily D proteins destined for peroxisomes have NH(2)-terminal hydrophilic region adjacent to TMS1. However, only P70R lacks the region and is translated with NH(2)-terminal hydrophobic TMS1. Furthermore, attachment of the NH(2)-terminal hydrophilic domain to the NH(2)-terminus of P70R excluded P70R from the ER-targeting pathway. These data suggest that P70R resides in the ER but not the peroxisomal membranes, and the hydrophobic property of NH(2)-terminal region determines the subcellular localization of ABC subfamily D proteins.