Background: The asparagine (N) to serine (S) substitution at codon 171 (N171S) of the prion protein (PrP) has been reported as a polymorphism with a prevalence of 5-8% in the African population. It has been also identified in a family with a prion-related psychiatric disorder. However, the association of the substitution with prion disease is far from being conclusive, because no pathological PrP (PrPSc) or prion-related neuropathological changes have been reported in subjects with N171S to date. Objectives: To report a Creutzfeldt-Jakob disease (CJD) case with N171S substitution. Methods: Approaches used include one and two dimensional SDS-PAGE and Western blotting, neurohistology, and prion protein gene (PRNP) analysis. Results: The patient was a 78-year old African-American male with the N171S substitution exhibited the clinical triad of dementia, myoclonus, and ataxia as well as a heterozygous N171S substitution, 129 Val/Val (V/V) polymorphism, and the silent 117alanine/alanine polymorphism. Fine spongiform degeneration was observed using H&E staining with intense synaptic PrP immunostaining as demonstrated by immunohistochemistry with the 3F4 antibody. Western blot analysis showed the presence of proteinase K (PK) resistant PrP (PrP27- 30) in the brain tissue. Compared to the PK-resistant PrP associated with sporadic CJD (sCJD), the monoglycosylated fragment in the subject is split into two bands, migrating at ~26 and 25 kDa, respectively. Furthermore, diglycosylated PrP was virtually undetectable and the migration of the non-glycosylated fragment is slower than that of sCJD VV1 control. Discussion: Our study suggests that the protein with N171S substitution may possess both a unique structure and changed glycans, which favors the hypothesis that this case represents a novel familial form of CJD rather than sporadic CJD. (Supported by the CJD Foundation, NIH NS062787, NIA AG-14359, NIH NS052319, and CDC UR8/CCU515004.)