The membrane integration of polytopic proteins is coordinated at the endoplasmic reticulum (ER) by the conserved Sec61 translocon, which facilitates the lateral release of transmembrane (TM) segments into the lipid phase during polypeptide translocation. Here we use a site-specific crosslinking strategy to study the membrane integration of a new model protein and show that the TM segments of the P2X2 receptor are retained at the Sec61 complex for the entire duration of the biosynthetic process. This extremely prolonged association implicates the Sec61 complex in the regulation of the membrane integration process, and we use both in vitro and in vivo analyses to study this effect further. TM-segment retention depends on the association of the ribosome with the Sec61 complex, and complete lateral exit of the P2X2 TM segments was only induced by the artificial termination of translation. In the event of the premature release of P2X2 TM1 from the ER translocon, the truncated polypeptide fragment was to found aggregate in the ER membrane, suggesting a distinct physiological requirement for the delayed release of TM segments from the ER translocon site.