The N-terminal polybasic region of the normal prion protein, PrPC, which encompasses residues 23-31, is important for prion pathogenesis by affecting conversion of PrPC into the pathogenic isoform, PrPSc. We previously reported transgenic mice expressing PrP with residues 25-50 deleted in the PrP-null background, designated as Tg(PrP∆preOR)/Prnp 0/0 mice. Here, we produced two new lines of Tg(PrP∆preOR)/Prnp 0/0 mice, each expressing the mutant protein, PrP∆preOR, 1.1 and 1.6 times more than PrPC in wild-type mice, and subsequently intracerebrally inoculated RML and 22L prions into them. The lower expresser showed slightly reduced susceptibility to RML prions but not to 22L prions. The higher expresser exhibited enhanced susceptibility to both prions. No prion transmission barrier was created in Tg(PrP∆preOR)/Prnp 0/0 mice against full-length PrPSc. PrPSc∆preOR accumulated in the brains of infected Tg(PrP∆preOR)/Prnp 0/0 mice less than PrPSc in control wild-type mice, although lower in RML-infected Tg(PrP∆preOR)/Prnp 0/0 mice than in 22L-infected mice. Prion infectivity in infected Tg(PrP∆preOR)/Prnp 0/0 mice was also lower than that in wild-type mice. These results indicate that deletion of residues 25-50 only slightly affects prion susceptibility, the conversion of PrPC into PrPSc, and prion infectivity in a strain-specific way. PrP∆preOR retains residues 23-24 and lacks residues 25-31 in the polybasic region. It is thus conceivable that residues 23-24 rather than 25-31 are important for the polybasic region to support prion pathogenesis. However, other investigators have reported that residues 27-31 not 23-24 are important to support prion pathogenesis. Taken together, the polybasic region might support prion pathogenesis through multiple sites including residues 23-24 and 27-31.