Although the protein-only hypothesis proposes that it is the conformation of abnormal prion protein (PrPSc) that determines strain diversity, the molecular basis of strains remains to be elucidated. In the present study, we generated a series of mutations in the normal prion protein (PrPC) in which a single glutamine residue was replaced with a basic amino acid and compared their abilities to convert to PrPSc in cultured neuronal N2a58 cells infected with either the Chandler or 22L mouse-adapted scrapie strain. In mice, these strains generate PrPSc of the same sequence but different conformations, as judged by infrared spectroscopy. Substitutions at codons 97, 167, 171, and 216 generated PrPC that resisted conversion and inhibited the conversion of coexpressed wild-type PrP in both Chandler-infected and 22L-infected cells. Interestingly, substitutions at codons 185 and 218 gave strain-dependent effects. The Q185R and Q185K PrP were efficiently converted to PrPSc in Chandler-infected but not 22L-infected cells. Conversely, Q218R and Q218H PrP were converted only in 22L-infected cells. Moreover, the Q218K PrP exerted a potent inhibitory effect on the conversion of coexpressed wild-type PrP in Chandler-infected cells but had little effect on 22L-infected cells. These results show that two strains with the same PrP sequence but different conformations have differing abilities to convert the same mutated PrPC.