The human UDP-glucuronosyltransferase isoform UGT1A6 is predicted to be a type I transmembrane protein anchored in the endoplasmic reticulum by a single C-terminal transmembrane domain, followed by a short cytoplasmic tail. This topology is thought to be established through the sequential action of a cleavable N-terminal signal peptide and of a C-terminal stop transfer/anchor sequence. We found that the deletion of the signal peptide did not prevent membrane targeting and insertion of this protein expressed in an in vitro transcription/translation system or in yeast Pichia pastoris. Interestingly, the same results were obtained when the protein was depleted of both the signal peptide and the C-terminal transmembrane domain/cytoplasmic tail sequences, suggesting the presence of an internal topogenic element able to translocate and retain UGT1A6 in the endoplasmic reticulum membrane in vitro and in yeast cells. To identify such a sequence, the insertion of several N-terminal deletion mutants of UGT1A6 into microsomal membranes was investigated in vitro. The data clearly showed that the deletion of the N-terminal end did not affect endoplasmic reticulum targeting and retention until residues 140-240 were deleted. The signal-like activity of the 140-240 region was demonstrated by the ability of this segment to confer endoplasmic reticulum residency to the cytosolic green fluorescent protein expressed in mammalian cells. Finally, we show that this novel topogenic sequence can posttranslationally mediate the translocation of UGT1A6. This study provides the first evidence that the membrane assembly of the human UGT1A6 involves an internal signal retention sequence.