Isoamyl monochloroacetate (i-AmOClAc) inhibited the growth of sake yeast. AOF10, which is resistant to isoamyl monofluoroacetate and has low esterase activity toward isoamyl acetate (i-AmOAc), also showed resistance to i-AmOC1Ac. Monochloroacetic acid formed by an esterase through the hydrolysis of i-AmOCLAc was more effective at glycolysis inhibition than i-AmOClAc. Forty five mutants resistant to i-AmOClAc were isolated from a parental haploid strain (HL-69) derived from sake yeast. Three had less than 20% of the original activity of the esterase. All mutants fermented sake mash as well as the parent strain, HL-69, and accumulated 1.3 to 1.7-fold the levels of i-AmOAc and 1.8 to 2.4-fold the levels of isobutyl acetate accumulated by HL-69. Surprisingly one mutant (CL-90), which accumulated 1.7-fold the amount of i-AmOAc and 1.3-fold the amount of ethyl acetate, exhibited increased alcohol acetyltransferase (EC 2.3.1.84) activity (1.6-fold). Esterase patterns were also examined by gel electrophoresis and activity staining. Changes in the intensity of the strongest band (EST2) were closely related to the activity of the esterase toward i-AmOAc.