Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC) respectively act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the non-selective interaction between the ribosome and the Sec61 complex. The Sec61 complex, serving as both the high affinity ribosome receptor and the translocation channel, is performing two very different functions which presumably requires different activity domains within the Sec61 complex. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the N and C-terminus of Sec61α was used to map the location of proteolysis cleavage sites. We observed a striking correlation between a loss of ribosome binding activity and the digestion of the C-terminal tail or cytoplasmic loop 8 of Sec61α. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether ribosome binding to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind ribosomes with high affinity at physiological ionic strength remain active in SRP-independent translocation indicating that ribosome binding and translocation promotion are separable activities of the Sec61 complex. Translocation promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.