Only few studies have explored the importance of functional diversity in temperate agricultural grasslands in relation to nitrogen (N) uptake. This study investigates the consequence of growing deep-rooted plants together with grass-clover mixtures in terms of N uptake efficiency from deep soil layers. The objective was to compare the N uptake of the shallow-rooted grassland species Lolium perenne and Trifolium repens; and the deep-rooted species Cichorium intybus and Medicago sativa in monocultures and mixtures. We hypothesized that growing deep-rooted plant species in mixture with shallow-rooted species increases the N uptake from deep soil layers partly through competition. A 15N tracer study was carried out with 15N enriched ammonium-sulphate placed at three different soil depths (40, 80 and 120 cm). To recover 15N, above-ground plant biomass was harvested after 10 days. We described the decline of 15N uptake with depth by using an exponential decay function. The studied plant communities showed the same relative decline in 15N uptake by increasing soil depths, but different capacities in total 15N uptake. Monoculture L. perenne foraged less 15N in all depths compared to the other four plant communities. The relative 15N uptake of individual plant species grown in mixture decreased stronger with depth than in monoculture. Thus, both findings rejected our hypothesis