During polytopic protein biogenesis, multiple transmembrane segments (TMs) must pass through the ribosome exit tunnel and into the Sec61 translocon prior to insertion into the endoplasmic reticulum membrane. To investigate how movement of a newly synthesized TM along this integration pathway might be influenced by synthesis of a second TM, we used photocrosslinking probes to detect proximity of ribosome-bound nascent polypeptides to Sec6. Probes were inserted at sequential sites within TM2 of the aquaporin-1 water channel by in vitro translation of truncated mRNAs. TM2 first contacted Sec61 when the probe was positioned ~38 residues from the ribosome peptidyltransferase center (PTC), and Sec61-TM2 photoadducts decreased markedly when the probe was more than 80 residues from the PTC. Unexpectedly, as nascent chain length was gradually extended, photocrosslinking at multiple sites within TM2 abruptly and transiently decreased, indicating that TM2 initially entered, withdrew, and then re-entered Sec61. This brief reduction in TM2 photocrosslinking coincided with TM3 synthesis. Replacement of TM3 with a secretory reporter domain or introduction of proline residues into TM3 changed the TM2 crosslinking profile and this biphasic behavior. These findings demonstrate that 1° and likely 2° structure of the nascent polypeptide within the ribosomal exit tunnel can influence the timing with which topogenic determinants contact, enter and pass through the translocon.