Efficient production of pyruvate from glucose by Torulopsis glabrata IFO 0005 was investigated. A large amount of pyruvate was accumulated in a practical semisynthetic fermentation medium which contained both ammonium sulfate and soybean hydrolyzate as nitrogen sources, instead of the Polypepton used in the previous study. A significant increase in the maximum amount of pyruvate accumulated was noted in a fed-batch culture in a jar fermenter. The fermentative conditions were optimized in an Erlenmeyer bask, and then in a jar fermentor in this study. Of the fermentative conditions, the concentrations of four vitamins (nicotinic acid, thiamine, pyridoxine and biotin) and aeration affected the pyruvate fermentative productivity. The optimum concentration of nicotinic acid was 8 mg/l, which was 20 times higher than that required for full growth. The optimum concentration of thiamine was 0.03 mg/l, which is 40% lower than that for full growth. Pyruvate production was markedly dependent on aeration. The maximum final accumulation of pyruvic acid was achieved under anaerobic conditions, but a greater yield of pyruvic acid was obtained under aerobic conditions. Based on the optimum conditions in an Erlenmeyer flask, the optimal initial and Feeding conditions in a jar fermenter were determined. Furthermore, we observed that cells grown under aerobic conditions developed mitochondria well compared with ones grown under anaerobic conditions, and investigated the effects of inhibitors, such as rotenone, of the electron transport chain, which exists in mitochondria, on pyruvate fermentation. The amount of pyruvate accumulated grown in the presence of 1.0 mM rotenone was 20% less than that accumulated grown in its absence by T. glabrata IFO 0005. Consequently, fed-batch fermentation with successive additions of glucose resulted in a maximum accumulation of 770 mM (67.8 g/l) pyruvic acid in 63 h (yield, 49.4%). The by-products were found to be 4 g/l of ethanol, 5 g/l of glycerol and 2 g/l of para-pyruvic acid.