The production of hydrogen sulfide (H2S) during yeast fermentation contributes negatively to wine aroma. We have mapped naturally occurring mutations in commercial wine strains that affect production of H2S. A dominant R(310)G mutant allele of MET2, which encodes homoserine O-acetyltransferase, is present in several wine yeast strains as well as in the main lab strain S288c. Reciprocal hemizygosity and allele swap experiments demonstrated that the MET2 (R310G) allele confers reduced H2S production. Mutations were also identified in genes encoding the two subunits of sulfite reductase, MET5 and MET10, which were associated with reduced H2S production. The most severe of these, an allele of MET10, showed five additional phenotypes: reduced growth rate on sulfate, elevated secretion of sulfite, and reduced production in wine of three volatile sulfur compounds: methionol, carbon disulfide and methylthioacetate. Alleles of MET5 and MET10, but not MET2, affected H2S production measured by colour assays on BiGGY indicator agar, but MET2 effects were seen when bismuth was added to agar plates made with Sauvignon blanc grape juice. Collectively, the data are consistent with the hypothesis that H2S production during wine fermentation results predominantly from enzyme activity in the sulfur assimilation pathway. Lower H2S production results from mutations that reduce the activity of sulfite reductase, the enzyme that produces H2S, or that increase the activity of l-homoserine-O-acetyltransferase, which produces substrate for the next step in the sulfur assimilation pathway.