The Pichia stipitis xylose reductase gene (XYL1) was inserted into an autonomous plasmid that P. stipitis maintains in multicopy. The plasmid pXOR with the XYL1 insert or a control plasmid pJM6 without XYL1 was introduced into P. stipitis. When grown on xylose under aerobic conditions, the strain with pXOR had up to 1.8-fold higher xylose reductase (XOR) activity than the control strain. Oxygen limitation led to higher XOR activity in both experimental and control strains grown on xylose. However, the XOR activities of the two strains grown on xylose were similar under oxygen limitation. When grown on glucose under aerobic or oxygen-limited conditions, the experimental strain had XOR activity up to 10 times higher than that of the control strain. Ethanol production was not improved, but rather it decreased with the introduction of pXOR compared to the control, and this was attributed to nonspecific effects of the plasmid.