The effect of water stress on respiration and mitochondrial electron transport has been studied in soybean (Glycine max) leaves, using the oxygen-isotope-fractionation technique. Treatments with three levels of water stress were applied by irrigation to replace 100%, 50%, and 0% of daily water use by transpiration. The levels of water stress were characterized in terms of light-saturated stomatal conductance (g(s)): well irrigated (g(s) > 0.2 mol H(2)O m(-2) s(-1)), mildly water stressed (g(s) between 0.1 and 0.2 mol H(2)O m(-2) s(-1)), and severely water stressed (g(s) < 0.1 mol H(2)O m(-2) s(-1)). Although net photosynthesis decreased by 40% and 70% under mild and severe water stress, respectively, the total respiratory oxygen uptake (V(t)) was not significantly different at any water-stress level. However, severe water stress caused a significant shift of electrons from the cytochrome to the alternative pathway. The electron partitioning through the alternative pathway increased from 10% to 12% under well-watered or mild water-stress conditions to near 40% under severe water stress. Consequently, the calculated rate of mitochondrial ATP synthesis decreased by 32% under severe water stress. Unlike many other stresses, water stress did not affect the levels of mitochondrial alternative oxidase protein. This suggests a biochemical regulation (other than protein synthesis) that causes this mitochondrial electron shift.