Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.