We have identified membrane components which are adjacent to type I and type II signal-anchor proteins during their insertion into the membrane of the ER. Using two different cross-linking approaches a 37-38-kD nonglycosylated protein, previously identified as P37 (High, S., D. Görlich, M. Wiedmann, T. A. Rapoport, and B. Dobberstein. 1991. J. Cell Biol. 113:35-44), was found adjacent to all the membrane inserted nascent chains used in this study. On the basis of immunoprecipitation, this ER protein was shown to be identical to the recently identified mammalian Sec61 protein. Thus, Sec61p is the principal cross-linking partner of both type I and type II signal-anchor proteins during their membrane insertion (this work), and of secretory proteins during their translocation (Görlich, D., S. Prehn, E. Hartmann, K.-U. Kalies, and T. A. Rapoport. 1992. Cell. 71:489-503). We propose that membrane proteins of both orientations, and secretory proteins employ the same ER translocation sites, and that Sec61p is a core component of these sites.