Background: The possible coexistence of different strains in natural prion isolates is the subject of enduring discussions and might have implications for prion strains surveillance. Nonetheless, this possibility has not yet been proved. In 2007, a case of ovine scrapie with features compatible with a mixed infection of classical scrapie and Nor98 was reported in Italy. Objectives: To characterize by bioassay the prion agent(s) involved in this unusual scrapie case. Methods: Previous studies showed that voles are susceptible to the prevalent Italian scrapie agent and resistant to Nor98, while tg338 mice show the contrary. Taking advantage of this differential susceptibility, we inoculated voles and tg338 with the brain stem (BS) and the cerebral cortex (CC) of the unusual case. Discriminatory WB showed a predominance of classical scrapie PrPres in BS, while CC mainly contained Nor98 PrPres. A classical scrapie isolate (ClS) from the same flock of the unusual case, as well as Nor98 isolates from Italy and Norway were used as controls. Survival times and attack rates were calculated in voles and tg338. Brains were analysed by discriminative WB for PrPSc type, by IHC and PET-blot for PrPSc deposition and by E&E for lesion profile. Results: Both CC and BS induced disease in tg338, with survival times similar to Nor98 controls. As expected, Tg338 were resistant to ClS control. In contrast, voles developed disease following inoculation of ClS control and BS, but not of CC and Nor98 controls. In voles, the neuropathological and molecular phenotypes upon infection with the unusual case were the same of those observed with ClS, while in Tg338 were identical to Nor98 controls. Discussion: Our results demonstrate that two independent and separable prion agents coexist in the isolate investigated. This implies that: - mixed prion infections can occur - classical scrapie and Nor98 are distinct entities - the isolation of prion strains from a mixture is affordable.