Impacts of elevated ground-level ozone (O₃) on nitrogen (N) uptake and allocation were studied on mature European beech (Fagus sylvatica L.) and Norway spruce (Picea abies [L.] Karst.) in a forest stand, hypothesizing that: (i) chronically elevated O₃ limits nutrient uptake, and (ii) beech responds more sensitively to elevated O₃ than spruce, as previously found for juvenile trees. Tree canopies were exposed to twice-ambient O₃ concentrations (2 × O₃) by a free-air fumigation system, with trees under ambient O₃ serving as control. After 5 years of O₃ fumigation, ¹⁵NH₄¹⁵NO₃ was applied to soil, and concentrations of newly acquired N (N_labelled) and total N (N_total) in plant compartments and soil measured. Under 2 × O₃, N_labelled and N_total were increased in the bulk soil and tended to be lower in fine and coarse roots of both species across the soil horizons, supporting hypothesis (i). N_labelled was reduced in beech foliage by up to 60, and by up to 50 in buds under 2 × O₃. Similarly, N_labelled in stem bark and phloem was reduced. No such reduction was observed in spruce, reflecting a stronger effect on N acquisition in beech in accordance with hypothesis (ii). In spruce, 2 × O₃ tended to favour allocation of new N to foliage. N_labelled in beech foliage correlated with cumulative seasonal transpiration, indicating impaired N acquisition was probably caused by reduced stomatal conductance and, hence, water transport under elevated O₃. Stimulated fine root growth under 2 × O₃ with a possible increase of below-ground N sink strength may also have accounted for lowered N allocation to above-ground organs. Reduced N uptake and altered allocation may enhance the use of stored N for growth, possibly affecting long-term stand nutrition.