Abstract Natural forms of prion diseases frequently originate by oral (p.o.) infection. However, quantitative information on the gastro-intestinal (GI) absorption of prions (i.e. the bioavailability and subsequent biodistribution) is mostly unknown. The main goal of this study was to evaluate the fate of prions after oral administration, using highly purified radiolabeled PrP Sc . The results showed a bi-phasic reduction of PrP Sc with time in the GI, except for the ileum and colon which showed sustained increases peaking at 3–6 hr, respectively. Plasma and whole blood 125 I-PrP Sc reached maximal levels by 30 min and 3 hr, respectively, and blood levels were constantly higher than plasma. Upon crossing the GI-tract 125 I-PrP Sc became associated to blood cells, suggesting that binding to cells decreased the biological clearance of the agent. Size-exclusion chromatography revealed that oligomeric 125 I-PrP Sc were transported from the intestinal tract, and protein misfolding cyclic amplification showed that PrP Sc in organs and blood retained the typical prion self-replicating ability. Pharmacokinetic analysis found the oral bioavailability of 125 I-PrP Sc to be 33.6%. Interestingly, 125 I-PrP Sc reached the brain in a quantity equivalent to the minimum amount needed to initiate prion disease. Our findings provide a comprehensive and quantitative study of the fate of prions upon oral infection.