Continuous fermentation using the industrial Saccharomyces cerevisiae diploid strain WW was carried out under acidic or high-temperature conditions to achieve acid- or thermo-tolerant mutants. Mutants isolated at pH 2.5 and 41 degrees C showed improved growth and fermentation ability under acidic and elevated temperature conditions. Haploid strains WW17A1 and WW17A4 obtained from the mutated diploid strain WW17A showed better growth and 4.5-6.5% higher ethanol yields at pH 2.7 than the original strains. Haploid strain WW12T4 obtained from mutated diploid strain WW12T showed 1.25-1.50 times and 2.8-4.7 times higher total cell number and cell viability, respectively, than the original strains at 42 degrees C. Strain AT, which had significantly improved acid- and thermo-tolerance, was developed by mating strain WW17A1 with WW12T4. Batch fermentation at 41 degrees C and pH 3.5 showed that the ethanol concentration and yield achieved during fermentation by strain AT were 55.4 g/L and 72.5%, respectively, which were 10 g/L and 13.4% higher than that of the original strain WW. The present study demonstrates that continuous cultivation followed by haploidization and mating is a powerful approach for enhancing the tolerance of industrial strains. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.