The EST2 gene, encoding an isoamyl acetate-hydrolyzing esterase, was disrupted in a diploid strain of Saccharomyces cerevisiae UT-1 (MATa/MAT alpha ura3/ura3 trp1/trp1 EST2/EST2), which is derived from the industrial sake yeast Kyokai no. 701 (strain K-701), by using two disruption plasmids (pDest2U, est2::URA3; and pDest2T, est2::TRP1) sequentially. Genomic Southern blot analysis revealed that both loci of the EST2 gene on the chromosome of strain UT-1 were disrupted. The resultant mutants were named UTUT-1 and UTUT-2 (MATa/MAT alpha ura3/ura3 trp1/trp1 est2::URA3/est2::TRP1). Deficiency in Est2p esterase was also confirmed by activity staining of the gel after native-polyacrylamide gel electrophoresis of cell extracts of the two mutant strains. Small scale sake brewing was carried out using these sake yeasts and the strains they were derived from, and their brewing properties were compared. The fermentation profiles of the four strains (strains K-701, UT-1, UTUT-1, and UTUT-2) were largely similar. The components of the resulting sake were also similar except for the acetate ester concentration, although strains UTUT-1 and UTUT-2 produced approximately 2-times more isoamyl acetate than the wild type K-701. These results strongly suggest that the EST2 gene product is likely to play a crucial role in the hydrolysis of isoamyl acetate in the sake mash. Strains UTUT-1 and UTUT-2, deficient in Est2p esterase, are suitable for sake brewing.