A universal feature of integral transmembrane proteins is a hydrophobic peptide segment that spans the lipid bilayer. These hydrophobic domains are important for terminating the translocation of the polypeptide chain across the membrane of the endoplasmic reticulum (a process termed stop transfer) and for integrating the protein into the bilayer. But a role for extracytoplasmic sequences in stop transfer and transmembrane integration has not previously been shown. Recently, a sequence which directs an unusual mode of stop transfer has been identified in the prion protein. This brain glycoprotein exists in two isoforms, which are identical both in primary amino-acid sequence and in containing phosphatidylinositol glycolipid linkages at their C termini, which can be cleaved by a phosphatidylinositol-specific phospholipase C9. But only one of the isoforms (PrPC) is released from cells on treatment with this phospholipase, indicating that the two isoforms have either different subcellular locations or transmembrane orientations. Consistent with this is the observation of two different topological forms in cell-free systems. An unusual topogenic sequence in the prion protein seems to direct these alternative topologies (manuscript in preparation). In the wheat-germ translation system, this sequence directs nascent chains to a transmembrane orientation; by contrast, in the rabbit reticulocyte lysate system, this sequence fails to cause stop transfer of most nascent chains. We have now investigated determinants in this unusual topogenic sequence that direct transmembrane topology, and have demonstrated that (1) a luminally disposed charged domain is required for stop transfer at the adjacent hydrophobic domain, (2) a precise spatial relationship between these domains is essential for efficient stop transfer, and (3) codons encompassing this hydrophilic extracytoplasmic domain confer transmembrane topology to a heterologous protein when engineered adjacent to the codons for a normally translocated hydrophobic domain. These results identify an unexpected functional domain for stop transfer in the prion protein and have implications for the mechanism of membrane protein biogenesis.