Background/Question/Methods Humans are indirectly increasing concentrations of surface ozone (O3) via fossil fuel combustion. Ozone is known to cause detrimental health impacts to biota, including reductions in crop yields, plant growth, and visible leaf injury. Less is known about the impacts of ozone on stomatal conductance and the concomitant impacts on transpiration. We investigated how ozone changes stomatal conductance and transpiration patterns using controlled open-top chamber experiments, focusing on the following questions: 1) How does short- and long-term exposure to elevated ozone influence stomatal behavior? 2) Do instantaneous and diurnal estimates of plant water loss differ under ozone exposure? and, 3) To what degree does ozone influence carbon assimilation independent of stomatal conductance? Results/Conclusions Exposure to short-term elevated ozone did not change instantaneous measures of stomatal conductance. However, after 12 weeks of exposure, daytime transpiration rates were significantly lower in plants exposed to zone. Further, although instantaneous measures suggested a decrease in conductance, diurnal patterns of stomatal behavior showed delayed responsiveness in stomata such that stomata closed later each day leading to larger daily water loss. Foliar d13C values tended to be higher for aspen seedlings exposed to elevated ozone compared to control seedlings, suggesting that stomata had a smaller aperture during the day. Carboxylation capacity, estimated using A-ci analysis, was lower in plants exposed to chronic ozone suggesting some level of damage independent of changes in stomatal function. Results of this study suggest that ozone causes a reduction in stomatal aperture during the day. However, stomata tend to remain open longer each day leading to increased daily transpiration.