The protein-only hypothesis predicts that infectious mammalian prions are composed solely of PrP, a misfolded conformer of the normal prion protein, PrP. However, to date, all wild type protein-only PrP preparations lack significant levels of prion infectivity. Using a systemic biochemical approach, our laboratory isolated and identified two different endogenous cofactor molecules, RNA (Deleault et al., 2003 [50]; Deleault et al., 2007 [59]) and phosphatidylethanolamine (Deleault et al., 2012 [61]; Deleault et al., 2012 [18]), which facilitate the formation of prions with high levels of specific infectivity, leading us to propose to the alternative hypothesis that cofactor molecules are required to form wild type infectious prions (Deleault et al., 2007 [59]; Deleault et al., 2012 [18]; Geoghegan et al., 2007 [57]). In addition, we found that purified cofactor molecules restrict the strain properties of chemically defined infectious prions (Deleault et al., 2012 [18]), suggesting a "cofactor selection" model in which natural variation in the distribution of strain-specific cofactor molecules in different parts of the brain may be responsible for strain-dependent patterns of neurotropism (Deleault et al., 2012 [18]; Geoghegan et al., 2007 [57]).