Protease‐resistant prion protein, total prion protein, and glial fibrillary acidic protein were measured in various brain regions from 9 subjects with fatal familial insomnia. Six were homozygotes methionine/methionine at codon 129 (mean duration, 10.7 ± 4 months) and 3 were heterozygotes methionine/valine (mean duration, 23 ± 11 months). In all subjects, protease‐resistant prion protein was detected in gray matter but not in white matter and peripheral organs. Its distribution was more widespread than that of the histopathological lesions, which were observed only in the presence of a critical amount of the abnormal protein. In the mediodorsal thalamic nucleus, however, a severe neuronal loss and astrogliosis were associated with relatively moderate amounts of protease‐resistant prion protein, suggesting a higher vulnerability. There was no overall correlation between amount of protease‐resistant prion protein and either glial fibrillary acidic protein or total prion protein. While protease‐resistant prion protein was virtually limited to subcortical areas and showed a selective pattern of distribution in the subjects with disease of the shortest duration, it was more widespread in the subjects with a longer clinical course, indicating that with time the disease process spreads within the brain. The kinetics of the accumulation of protease‐resistant prion protein varied among different brain regions: While in the neocortex and to a lesser extent in the limbic lobe and in the caudate nucleus, the amount increased with disease duration, in the mediodorsal thalamic nucleus and in the brainstem it was present in comparable amounts in all subjects regardless of the disease duration. These findings indicate that in fatal familial insomnia, the pathological phenotype is the result of the variability, in different brain regions, of the (1) timing and rate of accumulation of protease‐resistant prion protein, and (2) vulnerability to the presence of protease‐resistant prion protein.