Abstract Cultured brain slices rapidly replicate murine prions, exhibit prion pathology, and are amenable towards drug discovery, but have not been infected with human prions. As deer mice (Peromyscus maniculatus) are susceptible to human prions in vivo, here we investigated deer mouse organotypic brain slice cultures as a potential model of human prion disease. Deer mouse brain slices supported replication of rodent-adapted strains of scrapie and Creutzfeldt-Jakob disease (CJD), but they resisted infection with primary human prion inoculum. To better understand this discrepancy, we quantified prion replication rates, characterized cellular and molecular changes, and estimated inoculum clearance within wildtype CD1 and deer mouse brain slice cultures. Prion replication rates varied by species, strain, and brain region, independently of PrP sequence homology. Scrapie-infected CD1 cerebellar slice cultures exhibited the fastest prion replication rate, closely matching in vivo bioassay kinetics and showing neuronal and synaptic degeneration at similar timepoints. However, deer mouse slice cultures replicated deer mouse-adapted sCJD MM1 prions less efficiently than in vivo inoculation. These findings clarify both the utility and constraints of brain slice cultures in modeling prion disease and imply that the slice culture molecular environment may be suboptimal for human prion replication.