Human prion diseases are associated with a range of clinical presentations, and they are classified by both clinicopathological syndrome and etiology, with subclassification according to molecular criteria. Here, we describe procedures that are used within the MRC Prion Unit to determine a molecular diagnosis of human prion disease. Sequencing of the PRNP open reading frame to establish the presence of pathogenic mutations is described, together with detailed methods for immunoblot or immunohistochemical determination of the presence of abnormal prion protein in brain or peripheral tissues.