The signal peptidase complex (SPC) is a hetero-oligomeric membrane protein containing subunits of 12, 18, 21, 22/23, and 25 kDa. The 18- and 21-kDa subunits are mammalian homologs of SEC11 protein, which is necessary for signal peptide processing and cell viability in the yeast Saccharomyces cerevisiae. The functional and/or structural contributions of the 12-, 22/23-, and 25-kDa subunits to SPC activity have not yet been elucidated. To explore the structure of SPC subunits and their relationships to signal peptide processing and protein translocation, we have examined their endoplasmic reticulum (ER) membrane topology and biogenesis. Signal peptidase activity and SPC subunits are resistant to protease treatment in intact and detergent-solubilized membranes. Heat-denatured SPC subunits and SPC subunits translated in vitro are, however, protease sensitive, suggesting that the assembly of the oligomeric complex confers protease resistance. To define the membrane topology of SPC subunits, both wild-type subunits and subunit fusion proteins containing additional sites for N-linked glycosylation were assembled into microsomal membranes in vitro. Despite the presence of multiple hydrophobic domains, each subunit is anchored to the ER membrane by a single amino-terminal transmembrane domain in an Ncytoplasmic Cexoplasmic (type II) orientation. This topology places the bulk of the protein mass in the ER lumen and positions a putative serine-containing active site domain in SPC 18 and 21 at the same relative distance from the membrane as the analogous region in Escherichia coli leader peptidase. These studies have also revealed that, in spite of the temporal and perhaps physical association of the SPC with the process of protein translocation, SPC subunits integrate into the ER membrane by a signal recognition particle-dependent pathway and, hence, rely on the existence of a preformed translocation apparatus for their own membrane assembly.