The relationship between plant diversity and nitrate leaching into groundwater was investigated in a mid-European semi-natural grassland ecosystem. An experimental approach was used to directly manipulate plant diversity in the field, while holding other environmental factors constant. Species loss was simulated by establishing grassland communities of 16, 8, 4, 2, 1, and 0 plant species, composed of 3, 2, or I functional groups (grasses. legumes, and non-legume herbs). Every diversity treatment was replicated with several different species mixtures. Nitrate leaching was determined by continuous extraction of soil solution below the rooting zone and modeling of seepage rates. The concentration of nitrate in the soil solution was highly variable within each level of diversity. In bare ground plots and several low-diversity mixtures containing legumes, nitrate concentrations were higher than the official European Union threshold value for drinking water of 50 mg/L, with maximum values of up to 350 mg/L measured in Trifolium pratense monocultures. Total annual loss of nitrate was unaffected by the number of plant species or functional groups, but it was highly dependent on the specific species composition of the communities, and plots with legumes lost significantly more nitrate than plots without them. Aboveground biomass had no influence on nitrate loss, whereas leaching was negatively correlated with increasing root biomass. The abundance of legumes within a community, litter decomposition rates, and net nitrification were all positively correlated with total nitrate loss. However, in those communities containing legumes, leaching decreased with increasing diversity, because higher species richness led to a reduction in legume dominance, to a reduced nitrate supply through nitrification, and to a complementary uptake of nitrate by grasses and non-leguminous herbs. Based on these results, we expect that increasing the diversity of non-leguminous species or functional groups would reduce the risk of nitrate leaching in low-diversity grass-clover mixtures of ley-farming systems, while allowing for a more efficient exploitation of the beneficial fertilization effect provided by legumes