Variations in prions, which cause different incubation times and deposition patterns of the prion protein isoform called PrP(Sc), are often referred to as 'strains'. We report here a highly sensitive, conformation-dependent immunoassay that discriminates PrP(Sc) molecules among eight different prion strains propagated in Syrian hamsters. This immunoassay quantifies PrP isoforms by simultaneously following antibody binding to the denatured and native forms of a protein. In a plot of the ratio of antibody binding to denatured/native PrP graphed as a function of the concentration of PrP(Sc), each strain occupies a unique position, indicative of a particular PrP(Sc) conformation. This conclusion is supported by a unique pattern of equilibrium unfolding of PrP(Sc) found with each strain. Our findings indicate that each of the eight prion strains has a PrP(Sc) molecule with a unique conformation and, in accordance with earlier results, indicate the biological properties of prion strains are 'enciphered' in the conformation of PrP(Sc) and that the variation in incubation times is related to the relative protease sensitivity of PrP(Sc) in each strain.