A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrP(C)) into the scrapie isoform, denoted PrP(Sc). Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrP(C) and PrP(Sc); however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrP(C) is converted in scrapie-infected cells, suggesting that not all PrP(C) species are suitable substrates for the conversion. On the basis of the observation that PrP(C) can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrP(C), the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrP(C). Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrP(C) dimerization completely blocked its conversion into PrP(Sc) in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrP(C) dimers had a dominant-negative inhibition effect on the conversion of monomeric PrP(C). Our findings suggest that PrP(C) monomers are the major substrates for PrP(Sc) propagation and that it may be possible to halt prion formation by stabilizing PrP(C) dimers.