Several lines of evidence suggest that various cofactors may be required for prion replication. PrP binds to polyanions, and RNAs were shown to promote the conversion of PrP(C) into PrP(Sc) in vitro. In the present study, we investigated strain-specific differences in RNA requirement during in vitro conversion and the potential role of RNA as a strain-specifying component of infectious prions. We found that RNase treatment impairs PrP(Sc)-converting activity of 9 murine prion strains by protein misfolding cyclic amplification (PMCA) in a strain-specific fashion. While the addition of RNA restored PMCA conversion efficiency, the effect of synthetic polynucleotides or DNA was strain dependent, showing a different promiscuity of prion strains in cofactor utilization. The biological properties of RML propagated by PMCA under RNA-depleted conditions were compared to those of brain-derived and PMCA material generated in the presence of RNA. Inoculation of RNA-depleted RML in Tga20 mice resulted in an increased incidence of a distinctive disease phenotype characterized by forelimb paresis. However, this abnormal phenotype was not conserved in wild-type mice or upon secondary transmission. Immunohistochemical and cell panel assay analyses of mouse brains did not reveal significant differences between mice injected with the different RML inocula. We conclude that replication under RNA-depleted conditions did not modify RML prion strain properties. Our study cannot, however, exclude small variations of RML properties that would explain the abnormal clinical phenotype observed. We hypothesize that RNA molecules may act as catalysts of prion replication and that variable capacities of distinct prion strains to utilize different cofactors may explain strain-specific dependency upon RNA.