Impacts of elevated ground-level ozone (O(3)) 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 O(3) limits nutrient uptake, and (ii) beech responds more sensitively to elevated O(3) than spruce, as previously found for juvenile trees. Tree canopies were exposed to twice-ambient O(3) concentrations (2 × O(3)) by a free-air fumigation system, with trees under ambient O(3) serving as control. After 5 years of O(3) fumigation, (15)NH(4)(15)NO(3) 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 × O(3), 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 × O(3). 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 × O(3) 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 O(3). Stimulated fine root growth under 2 × O(3) 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.