The cleavage of signal sequences of secretory and membrane proteins by the signal peptidase complex occurs in the lumen of the endoplasmic reticulum. Mammalian signal peptidase consists of five subunits. Four have been cloned, SPC18, SPC21, SPC22/23, and SPC25, of which all but SPC25 have been demonstrated to be single-spanning membrane proteins exposed to the lumen of the endoplasmic reticulum. We have determined the cDNA sequence of the remaining 12-kDa subunit (SPC12) as well as the membrane topologies of SPC12 and SPC25 in rough microsomes. Both polypeptides span the membrane twice with their N and C termini facing the cytosol and contain only very small, if any, lumenal domains. Therefore, SPC12 and SPC25 are likely to be involved in processes other than the enzymatic cleavage of the signal sequence.