Methods enabling prion replication ex vivo are important for advancing prion studies. However, few such technologies exist, and many prion strains are not amenable to them. Here we describe a prion organotypic slice culture assay (POSCA) that allows prion amplification and titration ex vivo under conditions that closely resemble intracerebral infection. Thirty-five days after contact with prions, mouse cerebellar slices had amplified the abnormal isoform of prion protein, PrP^Sc, >10⁵-fold. This is quantitatively similar to amplification in vivo, but fivefold faster. PrP^Sc accumulated predominantly in the molecular layer, as in infected mice. The POSCA detected replication of prion strains from disparate sources, including bovines and ovines, with variable detection efficiency. Pharmacogenetic ablation of microglia from POSCA slices led to a 15-fold increase in prion titers and PrP^Sc concentrations over those in microglia-containing slices, as well as an increase in susceptibility to infection. This suggests that the extensive microglial activation accompanying prion diseases represents an efficacious defensive reaction.