We developed a novel co-culture system composed of two fermenters and two microfiltration modules for efficient ethanol production from a mixture of glucose and xylose by co-culture of Pichia stipitis and Saccharomyces cerevisiae, In a conventional co-culture where P. stipitis and S. cerevisiae were inoculated simultaneously into a single fermenter, it was impossible to maintain the specific oxygen uptake rate (qO(2)) at an optimum value for ethanol production from xylose by P. stipitis due to oxygen consumption by S. cerevisiae. The ethanol concentration obtained in the conventional co-culture was lower than that in a single culture of P. stipitis alone in which qO(2) was adjusted to an optimum level depending upon the kind of sugar consumed. The proposed co-culture system allowed regulation of the dissolved oxygen concentration at a level suitable for an individual yeast in each fermentor as well as the successful exchange of culture medium between two fermenters. When P. stipitis and S. cerevisiae were cultivated individually under different oxygen supply conditions in the new co-culture system, the yield and productivity of ethanol from a glucose and xylose mixture were higher than in single culture of P. stipitis alone.