Abnormal folding of the cellular prion protein (PrPC) is a key feature in prion diseases. Here we show that two pathogenic mutations linked to inherited prion diseases in humans severely affect folding and maturation of PrPC in the secretory pathway of neuronal cells. PrP-T183A and PrP-F198S adopt a misfolded and partially protease-resistant conformation, lack the glycosylphosphatidylinositol anchor, and are not complex glycosylated. These misfolded PrP mutants are not retained in the endoplasmic reticulum and are not subjected to the endoplasmic reticulum-associated degradation pathway. They rather are secreted, moreover, these mutants can be internalized by heterologous cells. Structural studies indicated that the side chains of Thr183 and Phe198 contribute to interactions between secondary structure elements in the C-terminal globular domain of PrPC. Consequently, we reasoned that a destabilized tertiary structure of these mutants could account for the defect in maturation. Indeed, mutations predicted to interfere selectively with the packing of the hydrophobic core of PrPC prevented the addition of the glycosylphosphatidylinositol anchor. Our study reveals that formation of the C-terminal globular domain of PrPC has an impact on membrane anchoring and indicates that misfolded secreted forms of the prion protein are linked to inherited prion diseases in humans.