The NH2-terminal fragments of sterol regulatory element-binding proteins (SREBPs) are released from endoplasmic reticulum membranes by proteases whose activities depend upon SREBP cleavage-activating protein (SCAP), a polytopic endoplasmic reticulum membrane protein. The activity of SCAP is inhibited by sterols, which appear to interact with the polytopic membrane domain of SCAP. Here, we use protease protection and N-linked glycosylation site-mapping techniques to define the topology of the eight membrane-spanning domains of SCAP. The data indicate that the NH2 terminus and COOH terminus of SCAP face the cytosol. The long intralumenal loops after membrane-spanning segments 1 and 7 are glycosylated, confirming their lumenal location. The region comprising membrane-spanning segments 2-6 shows sequence resemblance to putative sterol-sensing domains in three other proteins: 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), the Niemann-Pick C1 protein, and the morphogen receptor Patched. The orientation of the eight membrane-spanning segments in SCAP is consistent with the model proposed for HMG-CoA reductase (Olender, E. H., and Simoni, R. D. (1992) J. Biol. Chem. 267, 4223-4235). The membrane-spanning domains of SCAP and HMG-CoA reductase confer sterol sensitivity upon the functional activities of the two molecules. The common membrane topology of the two proteins is consistent with the notion that sterols regulate both proteins by a common mechanism.