Prion diseases are neurodegenerative diseases associated with the accumulation of a pathogenic isoform of the host-encoded prion protein. The cellular responses to prion infection are not well defined. By performing microarray analysis on cultured neuronal cells infected with prion strain 22L, in the group of up-regulated genes we observed predominantly genes of the cholesterol pathway. Increased transcript levels of at least nine enzymes involved in cholesterol synthesis, including the gene for the rate-limiting hydroxymethylglutaryl-CoA reductase, were detected. Up-regulation of cholesterogenic genes was attributable to a prion-dependent increase in the amount and activity of the sterol regulatory element-binding protein Srebp2, resulting in elevated levels of total and free cellular cholesterol. The up-regulation of cholesterol biosynthesis appeared to be a characteristic response of neurons to prion challenge, as cholesterogenic transcripts were also elevated in persistently infected GT-1 cells and prion-exposed primary hippocampal neurons but not in microglial cells and primary astrocytes. These results convincingly demonstrate that prion propagation not only depends on the availability of cholesterol but that neuronal cells themselves respond to prions with specific up-regulation of cholesterol biosynthesis.