A prion is an infectious protein, a concept derived from studies of mammalian spongiform encephalopathies. We discovered that the yeast Saccharomyces cerevisiae can be infected with two prions, two non-chromosomal genetic elements whose properties are those predicted for an infectious protein, not a nucleic acid replicon or a virus. The first, [URE3] is an altered form of Ure2p, the protein product of the chromosomal URE2 gene important in regulation of nitrogen metabolism. The second, [PSI], is an altered form of Sup35p, a subunit of the translation release factor and product of the chromosomal SUP35 gene. We find that Ure2p is more resistant to protease digestion in [URE3] strains than in wild-type strains, supporting the prion model for [URE3]. This protease resistance is not a constant concomitant of derepression of nitrogen metabolism. The N-terminal 65 aminoacid residues of Ure2p is sufficient to propagate [URE3], in the complete absence of the C-terminal nitrogen regulation domain. The overexpression of the URE2 protein, and not the URE2 RNA, is what induces the de novo formation of [URE3]. The C-terminal nitrogen regulation domain is only inactivated when covalently attached to the N-terminal domain, showing that [URE3] is propagated by interactions between the N-terminal prion domains. We have purified Ure2p to near homogeneity and have begun to characterize the difference between the normal and prion forms. Certain deletions within the C-terminal nitrogen regulation domain of Ure2p result in a 100-fold increase the frequency with which the [URE3] prion arises. In fact, either of two non-overlapping parts of Ure2p can, when overexpressed, induce the de novo appearance of [URE3].