A distinct conformational transition from the α-helix-rich cellular prion protein (PrP^C) into its β-sheet-rich pathological isoform (PrP^Sc) is the hallmark of prion diseases, a group of fatal transmissible encephalopathies that includes spontaneous and acquired forms. Recently, a PrP^Sc-like intermediate form characterized by the formation of insoluble aggregates and protease-resistant PrP species termed insoluble PrP^C (iPrP^C) has been identified in uninfected mammalian brains and cultured neuronal cells, providing new insights into the molecular mechanism(s) of these diseases. Here, we explore the molecular characteristics of the spontaneously formed iPrP^C in cultured neuroblastoma cells expressing wild-type or mutant human PrP linked to two familial prion diseases. We observed that although PrP mutation at either residue 183 from Thr to Ala (PrP^T183A) or at residue 198 from Phe to Ser (PrP^F198S) affects glycosylation at both N-linked glycosylation sites, the T183A mutation that results in intracellular retention significantly increased the formation of iPrP^C. Moreover, while autophagy is increased in F198S cells, it was significantly decreased in T183A cells. Our results indicate that iPrP^C may be formed more readily in an intracellular compartment and that a significant increase in PrP^T183A aggregation may be attributable to the inhibition of autophagy.