Abstract We investigated the membrane topogenesis of glucose-6-phosphatase (G6Pase), a multispanning membrane protein, on the endoplasmic reticulum. In COS-7 cells, the first transmembrane segment (TM1) with weak hydrophobicity is inserted into the membrane in the N-terminus-out/C-terminus-cytoplasm orientation. The following TM2 is inserted depending on TM3. TM3 has the same orientation as TM1. In contrast to data from living cells, the full-length molecule and N-terminal fusion constructs were not inserted into the membrane in a cell-free system. Addition of a signal recognition particle did not improve G6Pase insertion. When the 37-residue N-terminal segment was deleted, however, TM2 and TM3 were correctly inserted. We concluded that the three N-terminal TM segments are inserted into the membrane dependent on the two signal-anchor sequences of TM1 and TM3. TM1 is likely to be an unconventional signal sequence that barely functions in vitro. The 37-residue N-terminal segment inhibits the signal function of the following TM3 in cell-free systems.