Synthetic prions were produced in our laboratory by using recombinant mouse prion protein (MoPrP) composed of residues 89-230. The first mouse synthetic prion strain (MoSP1) was inoculated into transgenic (Tg) 9949 mice expressing N-terminally truncated MoPrP(Δ23-88) and WT FVB mice expressing full-length MoPrP. On first and second passage in Tg9949 mice, MoSP1 prions caused disease in 516 ± 27 and 258 ± 25 days, respectively; numerous, large vacuoles were found in the brainstem and gray matter of the cerebellum. MoSP1 prions passaged in Tg9949 mice were inoculated into FVB mice; on first and second passage, the FVB mice exhibited incubation times of 154 ± 4 and 130 ± 3 days, respectively. In FVB mice, vacuolation was less intense but more widely distributed, with numerous lesions in the hippocampus and cerebellar white matter. This constellation of widespread neuropatho-logic changes was similar to that found in FVB mice inoculated with Rocky Mountain Laboratory (RML) prions, a strain derived from a sheep with scrapie. Conformational stability studies showed that the half-maximal GdnHCl (Gdn_1/2) concentration for denaturation of MoSP1 prions passaged in Tg9949 mice was ≈4.2 M; passage in FVB mice reduced the Gdn_1/2 value to ≈1.7 M. RML prions passaged in either Tg9949 or FVB mice exhibited Gdn_1/2 values of ≈1.8 M. The incubation times, neuropathological lesion profiles, and Gdn_1/2 values indicate that MoSP1 prions differ from RML and many other prion strains derived from sheep with scrapie and cattle with bovine spongiform encephalopathy.