In CYH2/cyh2 heterozygous diploids of the yeast Saccharomyces cerevisiae resistance is dominant over sensitivity at low (0.5–5 μg/ml) cycloheximide (cyh) concentrations. The cyh-resistant haploid strain MMY1 confers relatively high (10 μg/ml) cyh-resistance to heterozygous diploids constructed by mating this strain with cyh-sensitive haploid strains. We present here a genetic and biochemical study of strain MMY1. Analysis of tetrads obtained from a MMY1 heterozygous diploid showed that two unlinked nuclear mutations, determining high-and low-cycloheximide resistance, were present in MMY1. From a genomic library of this strain, constructed in vector YCp50, two plasmids (pRC1 and pRC13) have been isolated which, respectively, confer high-and low-resistance phenotypes to cyh-sensitive S. cerevisiae strains. The restriction maps of pRC1 and pRC13 are totally unrelated. This finding suggests that the genes harboring the two mutations encoding cyh-resistance from MMY1 were cloned in plasmids pRC1 and pRC13, respectively. Pulse field gel electrophoresis showed that the DNA insert of pRC1 maps at either chromosome VII or XV, whereas that from pRC13 maps at chromosome XI. This latter gene appears to define a previously unreported locus and has been named cyh5. By restriction and nucleotide sequencing analysis, the cyh gene present in pRC1 has been shown to correspond to cyh2, which maps at chromosome VII. These results suggest that the dominant cyh-resistance phenotype conferred by MMY1 in heterozygous diploids is promoted by the presence of both cyh2 and cyh5. A 2.3 kb NcoI fragment, containing cyh2 from pRC1, has been inserted in vectors YCp50, YEp13 and YIp5. By selecting at discriminating cyh concentrations, the resulting constructs efficiently transform a variety of haploid and diploid yeasts of both laboratory and industrial origin.