In individuals with experimentally induced or naturally acquired TSEs prion infectivity and PrPTSE predominantly accumulate in the CNS but were also found, to varying degrees, in peripheral tissues. Together with CJD and scrapie CWD is one of three naturally occurring forms of prion disease. CWD is contagious and has shown an increasing spread R-7050 during the past few years in captive as well as free ranging cervids in North America. This has raised concerns for risks of zoonotic CWD transmission to humans via foodstuffs from cervids �C not least because the question of whether CWD prions have the ability to infect humans has not yet been definitely resolved. Skeletal muscles from farm- and game animals provide a frequent component of the human diet, and muscle tissue from scrapieinfected mice, hamsters and sheep, or from humans with classical or variant CJD has been previously shown to contain TSE infectivity and/or PrPTSE. In 2006, Angers et al. reported the detection of prion infectivity in specimens of skeletal muscle from CWD-affected mule deer by bioassay. Although such muscle samples induced disease in reporter animals, PrPTSE or its proteinaceous seeding activity could not be detected directly in skeletal muscles of CWD-infected cervids so far. Therefore, we applied a high-yield method for the extraction of PrPTSE from muscle samples of CWD-infected WTD and screened the extracts from different skeletal muscles by highly sensitive Western blotting for the presence of PrPTSE. In addition to farmed WTD culled in Saskatchewan, Canada, we also analyzed hunter-killed game WTD from Wisconsin, USA. All farmed and free-ranging WTD examined in our study had been officially diagnosed Ac-YVAD-pNA positive for CWD infection by the responsible authorities based on an analysis of brain and/or lymphatic tissue for the presence of PrPTSE, although no clinical signs of CWD had been recognized in these animals. In our study, we detected, for the first time, PrPTSE and PrPTSE-associated seeding activity in skeletal muscles of farmed and free-ranging cervids infected with CWD. Tissue-blotting revealed that PrPTSE was located in muscleassociated nerve fascicles while PrPTSE was not detectable in mycoytes.