Biosynthesis of the prion protein at the endoplasmic reticulum generates multiple topological forms. The topology of an individual chain is determined first by the localization of the N terminus and then by potential integration of the transmembrane domain into the lipid bilayer. Here, we provide the first evidence that signal sequences affect the latter of these events by demonstrating that some but not other signal sequences and signal sequence mutations result in significant increases in the fraction of prion protein nascent chains that integrate into the lipid bilayer. Through analysis of the prolactin signal sequence, an especially poor integration effector, we find that the N terminal and hydrophobic regions of the signal sequence affect integration most significantly. Mutations in either region result in a considerable increase in the number of chains that integrate. The effect of the signal sequence cannot be attributed to timing of signal cleavage or the state of the ribosome membrane junction, parameters previously found to affect protein biogenesis. We also present evidence that signal sequences that are poor integration effectors can promote integration under experimental conditions that allow the nascent chain more time to integrate. These findings reveal a previously unappreciated relationship between signal sequences and transmembrane integration.