Secretory proteins are segregated from cytosolic proteins by their translocation into the endoplasmic reticulum (ER). A modified secretory protein trapped during translocation across the ER membrane can be crosslinked to two previously identified proteins, Sec6lp and BiP (Kar2p). The dependence of this cross-linking upon proteins and small molecules was examined. Mutations in SEC62 and SEC63 decrease the ability of Sec61 p to be cross-linked to the secretory polypeptide trapped in translocation. ATP is also required for Interaction of Sec61 p with the secretory protein. Three kar2 alleles display defective translocation in vitro. Two of these alleles also decrease the ability of Sec6lp to be cross-linked to the secretory protein. The third allele, while exhibiting a severe translocation defect, does not affect the interaction of Sec61 p with the secretory protein. These results suggest that Sec6lp is directly involved in translocation and that BiP acts at two stages of the translocation cycle.