Conformational differences in abnormal prion proteins (PrP(Sc)) have been postulated to produce different prion phenotypes. During the interspecies transmission of prions, the conformation of PrP(Sc) may change with passage; however, little is known about the mechanism of PrP(Sc) transition. In this study, novel PrP(Sc)-specific monoclonal antibodies (mAbs) were developed that could detect the PrP(Sc) of mouse but not that of sheep. By using these mAbs, we attempted to examine PrP(Sc) accumulated in mice inoculated with sheep scrapie serially up to five passages. The presence of PrP(Sc) in the mice was confirmed at all passages; however, mAb-bound PrP(Sc) conformer was detected only from the third passage onward. The generated mAb enabled tracing of a particular conformer during adaptation in sheep-to-mice transmission of prion, suggesting that the conformational transition of PrP(Sc) was caused by propagation of this conformer. Such mAbs capable of discriminating conformational differences may allow us to address questions concerning PrP(Sc) conformation and strain diversity.