Although the protein-only hypothesis proposes that it is the conformation of abnormal prion protein (PrP^Sc) 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 (PrP^C) in which a single glutamine residue was replaced with a basic amino acid and compared their abilities to convert to PrP^Sc in cultured neuronal N2a58 cells infected with either the Chandler or 22L mouse-adapted scrapie strain. In mice, these strains generate PrP^Sc of the same sequence but different conformations, as judged by infrared spectroscopy. Substitutions at codons 97, 167, 171, and 216 generated PrP^C 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 PrP^Sc 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 PrP^C.