Over the years, a series of trembling aspen (Populus tremuloides Michx.) clones differing in O3 sensitivity have been identified from OTC studies. Three clones (216 and 271[(O3 tolerant] and 259 [O3 sensitive]) have been characterized for O3 sensitivity by growth and biomass responses, foliar symptoms, gas exchange, chlorophyll content, epicuticular wax characteristics, and antioxidant production. In this study we compared the responses of these same clones exposed to O3 under field conditions along a natural O3 gradient and in a Free-Air CO2 and O3 Enrichment (FACE) facility. In addition, we examined how elevated CO2 affected O3 symptom development. Visible O3 symptoms were consistently seen (5 out of 6 years) at two of the three sites along the O3 gradient and where daily one-hour maximum concentrations were in the range of 96 to 125 ppb. Clonal differences in O3 sensitivity were consistent with our OTC rankings Elevated CO2 (200 ppm over ambient and applied during daylight hours during the growing season) reduced visible foliar symptoms for all three clones from 31 to 96% as determined by symptom development in elevated O3 versus elevated O3 + CO2 treatments. Degradation of the epicuticular wax surface of all three clones was found at the two elevated O3 gradient sites. This degradation was quantified by a coefficient of occlusion which was a measure of stomatal occlusion by epicuticular waxes. Statistically significant increases in stomatal occlusion compared to controls were found for all three clones and for all treatments including elevated CO2, elevated O3, and elevated CO2 + O3. Our results provide additional evidence that current ambient O3 levels in the Great Lakes region are causing adverse effects on trembling aspen. Whether or not elevated CO2 in the future will alleviate some of these adverse effects, as occurred with visible symptoms but not with epicuticular wax degradation, is unknown.