The yeast Saccharomyces cerevisiae genome encodes several proteins that, in laboratory strains, can take up a stable, transmissible prion form. In each case, this requires the Asn/Gln-rich prion-forming domain (PrD) of the protein to be intact. In order to further understand the evolutionary significance of this unusual property, we have examined four different prion genes and their corresponding PrDs, from a number of naturally occurring strains of S. cerevisiae. In 4 of the 16 strains studied we identified a new allele of the SUP35 gene (SUP35Δ19) that contains a 19-amino-acid deletion within the N-terminal PrD, a deletion that eliminates the prion property of Sup35p. In these strains a second prion gene, RNQ1, was found to be highly polymorphic, with eight different RNQ1 alleles detected in the six diploid strains studied. In contrast, for one other prion gene (URE2) and the sequence of the NEW1 gene encoding a PrD, no significant degree of DNA polymorphism was detected. Analysis of the naturally occurring alleles of RNQ1 and SUP35 indicated that the various polymorphisms identified were associated with DNA tandem repeats (6, 12, 33, 42 or 57 bp) within the coding sequences. The expansion and contraction of DNA repeats within the RNQ1 gene may provide an evolutionary mechanism that can ensure rapid change between the [PRION+] and [prion–] states.