We have used the natural N‐glycosylation site in the N‐tail of cig30, a eukaryotic polytopic membrane protein, as a marker for N‐tail translocation across the microsomal membrane. Analysis of C‐terminally truncated cig30 constructs reveals that the first transmembrane segment is sufficient for translocation of the wild‐type N‐tail; in contrast, in a mutant with four arginines introduced into the N‐tail the second transmembrane segment is also required for efficient N‐tail translocation. Our observations imply a non‐sequential assembly mechanism in which the ultimate location of the N‐tail relative to the membrane may depend on more than one transmembrane segment.