ROT1 is an essential gene that has been related to cell wall biosynthesis, the actin cytoskeleton and protein folding. In order to help to understand its molecular function, we carried out a characterization of the Rot1 protein. It is primarily located at the endoplasmic reticulum–nuclear membrane facing the lumen. Rot1 migrates more slowly than expected, which might suggest post‐translational modification. Our results indicate that Rot1 is a protein that is neither GPI‐anchored nor O‐glycosylated. In contrast, it is N‐glycosylated. By a directed mutagenesis of several Asn residues, we identified that the protein is simultaneously glycosylated at N103, N107 and N139. Although the mutation of these three N sites is not lethal, cellular growth is impaired. Sequence analysis predicts a transmembrane domain at the C‐terminus. This fragment affects neither the targeting of the Rot1 protein to the ER nor its N‐glycosylation, although it is important for the anchoring of the protein to the membrane and for its functionality. The existence of a signal sequence at the N‐terminus has been suggested. However, deletion of this fragment impedes neither translocation to the ER nor N‐glycosylation, but it is required for cell viability. Finally, we found that Rot1 is translocated to the ER by an SRP‐independent post‐translational mechanism which depends on Sec62. Copyright © 2007 John Wiley & Sons, Ltd.