We investigated the effect of leaf age on the response of net photosynthesis (A), stomatal conductance ( g wv ), foliar injury, and leaf nitrogen concentration (N L) to tropospheric ozone (O 3) on Prunus serotina seedlings grown in open-plots (AA) and open-top chambers, supplied with either carbon-filtered or non-filtered air. We found significant variation in A, g wv , foliar injury, and N L ( P < 0.05) among O 3 treatments. Seedlings in AA showed the highest A and g wv due to relatively low vapor pressure deficit (VPD). Older leaves showed significantly lower A, g wv , N L, and higher foliar injury ( P < 0.001) than younger leaves. Leaf age affected the response of A, g wv , and foliar injury to O 3. Both VPD and N L had a strong influence on leaf gas exchange. Foliar O 3-induced injury appeared when cumulative O 3 uptake reached 8–12 mmol m −2, depending on soil water availability. The mechanistic assessment of O 3-induced injury is a valuable approach for a biologically relevant O 3 risk assessment for forest trees. Ozone effects on symptom development and leaf gas exchange interacted with leaf age and N-content on black cherry seedlings.