In addition to the familiar possibility of epistasis between nuclear loci, interactions may evolve between the mitochondrial and nuclear genomes in eukaryotic cells. We looked for such interactions in Saccharomyces cerevisiae genotypes evolved independently and asexually in the laboratory for 2000 generations, and in an ecologically distinct pathogenic S. cerevisiae strain. From these strains we constructed derivatives entirely lacking mitochondrial DNA and then used crosses to construct matched and unmatched pairings of nuclear and mitochondrial genomes. We detected fitness effects of such interactions in an evolved laboratory strain and in crosses between the laboratory and pathogen strains. In both cases, there were significant contributions to progeny fitness of both nuclear and mitochondrial genomes and of their interaction. A second evolved genotype showed incompatibility with the first evolved genotype, but the nuclear and mitochondrial contributions to this incompatibility could not be resolved. These results indicate that cytonuclear interactions analogous to those already known from plants and animals can evolve rapidly on an evolutionary timescale.