Ozone is a common ground-level air pollutant that can be harmful to human health, vegetation, and ecosystems. Ailanthus altissima (Tree-of-Heaven) is an exotic, invasive tree species found across most of the US that has been suggested as a bioindicator to detect phytotoxic levels of ambient ozone. We conducted greenhouse exposures and field studies to evaluate the ozone-sensitivity of Tree-of-Heaven, including seedlings from several seed sources. Severity of ozone-induced foliar symptoms was directly related to ozone concentration and varied among seed sources. Tree-of-Heaven seedlings out-planted in the field developed foliar symptoms induced by ambient ozone levels at or near the national ambient air quality standard (NAAQS) for photochemical oxidants and ozone. Our results provide data that may be considered when setting new a NAAQS for photochemical oxidants and ozone.