To learn about the molecular mechanism of protein translocation across the membrane of the endoplasmic reticulum (ER), the environment of nascent chains during the translocation process has been characterized using a variety of crosslinking approaches. These techniques have led to the identification of several proteins that interact transiently with the newly synthesized protein in the cytosol, during its passage across the membrane of the ER and in the ER lumen. Furthermore, lipids have been found to be in contact with membrane-inserted nascent chains, suggesting that the polypeptide enters the membrane in a protein-lipid interface.