Prion protein (PrP) is the only known constituent of the agents (called prions) that cause fatal neurodegenerative diseases in animals and humans. PrP derives from a host protein encoded by a single copy gene having three known exons in mice, cattle and sheep but only two exons in hamsters and humans. We have identified and sequenced the missing exon from the hamster PrP gene. The new hamster PrP exon is 83% identical to mouse exon 2 and 76% identical to exon 2 from cattle and sheep. PrP mRNAs containing the new exon 2 (mRNA[1+2+3]) were expressed in the colliculi, frontal cortex and hippocampus of normal hamsters at approximately 30% to approximately 50% of the levels of the mRNA without exon 2 (mRNA[1+3]). Expression of PrP mRNA[1+2+3] was increased in the colliculi beginning 49 days after inoculation with scrapie prions and reached a level 2.5 times normal by day 77. Increased expression of PrP mRNA[1+2+3] in the colliculi correlated with expression of glial fibrillary acidic protein (GFAP) mRNA. Expression of GFAP and PrP mRNAs was not significantly increased in the hippocampus or the frontal cortex during the disease. Our study shows that exon 2 plays a role in regulating the cellular expression of hamster PrP and suggests that mRNA[1+2+3] may be preferentially expressed in hamster astrocytes.