Three 80- to 95-month-old Holstein dairy cattle infected naturally with the agent of bovine spongiform encephalopathy (BSE) and slaughtered at abattoirs in Japan were examined for the distribution of disease-specific and protease-resistant prion protein (PrP(Sc)) by immunohistochemistry (IHC) and Western blot (WB) analyses. The cattle showed no clinical signs or symptoms relevant to BSE but were screened as positive by enzyme-linked immunosorbent assay, a rapid test for BSE. This positive result was confirmed by IHC or WB in a specimen of the medulla oblongata. Histopathologically, these cattle showed no vacuolation in tissue sections from the central nervous system except for the medulla oblongata. Both IHC and WB analyses revealed PrP(Sc) accumulation in the brain, spinal cord, satellite and ganglionic cells of the dorsal root ganglia, and the myenteric plexus of the distal ileum. In addition, small amounts of PrP(Sc) were detected in the peripheral nerves of 2 cattle by WB. No PrP(Sc) was demonstrated by either method in the Peyer's patches of the distal ileum; lymphoid tissues including the palatine tonsils, lymph nodes, and spleen; or other tissues. The distribution of PrP(Sc) accumulation in the preclinical stage was different between naturally infected cattle and cattle inoculated experimentally with the BSE agent.