With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt–Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrP^Sc) used limited proteolysis to digest the precursor cellular PrP (PrP^C). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrP^Sc, we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrP^Sc in all 10–24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, ≈90% of the total PrP^Sc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrP^C. Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.