During protein integration into the endoplasmic reticulum, the N-terminal domain preceding the type I signal-anchor sequence is translocated through a translocon. By fusing a streptavidin-binding peptide tag to the N terminus, we created integration intermediates of multispanning membrane proteins. In a cell-free system, N-terminal domain (N-domain) translocation was arrested by streptavidin and resumed by biotin. Even when N-domain translocation was arrested, the second hydrophobic segment mediated translocation of the downstream hydrophilic segment. In one of the defined intermediates, two hydrophilic segments and two hydrophobic segments formed a transmembrane disposition in a productive state. Both of the translocating hydrophilic segments were crosslinked with a translocon subunit, Sec61alpha. We conclude that two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. These observations indicate the remarkably flexible nature of the translocon.