The phenotype of human sporadic prion diseases is affected by patient genotype at codon 129 of the prion protein (PrP) gene, the site of a common methionine/valine polymorphism, and by the type of the scrapie PrP (PrP Sc), which likely reflects the prion strain. However, two distinct disease phenotypes, identified as sporadic Creutzfeldt-Jakob disease (M/M2 sCJD) and sporadic fatal insomnia (sFI), share methionine homozygosity at codon 129 and PrPSc type 2. One-dimensional gel electrophoresis and immunoblotting reveal no difference between the M/M2 sCJD and sFI species of PrPSc in gel mobility and glycoform ratio. In contrast, the two-dimensional immunoblot demonstrates that in M/M2 sCJD the full-length PrPSc form is overrepresented and carries glycans that are different from those present in the PrPSc of sFI. Because the altered glycans are detectable only in the PrPSc and not in the normal or cellular PrP (PrPC), they are likely to result from preferential conversion to PrPSc of rare PrPC glycoforms. This is the first evidence that a qualitative difference in glycans contributes to prion diversity.