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, 15NH4 15NO3 was applied to soil, and concentrations of newly acquired N (Nlabelled) and total N (Ntotal) in plant compartments and soil measured. Under 2 × O3, N labelled and Ntotal 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). Nlabelled was reduced in beech foliage by up to 60, and by up to 50 in buds under 2 × O3. Similarly, Nlabelled 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. Nlabelled 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.