Eighteen mutants resistant to 5',5',5'-trifluoroleucine (TFL), a leucine analog, were isolated in Saccharomyces cerevisiae strains YNN281 and YNN282. The mutants were characterized genetically and clustered in two groups, one comprising all the dominant (TFL1) and the other one all the recessive (tfl2) mutations. Genetic and biochemical data suggested that the dominant mutations are located on the LEU4 gene, coding for alpha-isopropylmalate synthase I. These mutations resulted in accumulation of leucine as a consequence of the synthesis of an enzyme insensitive to the feedback inhibition by leucine. Leucine excretion in the TFL1 mutants appeared to be affected by the genetic background of the strain and was greatly influenced by lysine metabolism. The measurement of intra- and extracellular amino acid concentrations in prototrophic strains carrying TFL1 or tfl2 genes showed that both were leucine overproducers. Some of the TFL-resistant mutants were tested in alcoholic fermentation of grape must: analysis of the fermentation secondary metabolites showed that the major effect of the TFL-resistant strains was an increased production of isoamyl alcohol compared to that of the parental strain.