Symbiotic N-2 fixation may be an important source of N for legumes in alpine ecosystems, though, this has hardly been investigated. Symbiotic N-2 fixation in nine legume species in permanent grassland over an altitudinal gradient (from 900 up to 2600 m a.s.l.) was investigated in the Swiss Alps on strictly siliceous soils. To assess symbiotic N-2 fixation, an enriched N-15 isotope dilution method was established for low N input, permanent grasslands and was evaluated with the N-15 natural abundance method. The non-N-2-fixing reference species used in both methods differed significantly in their N-15 atom%-excess. However, when several reference species were combined, the enriched N-15 isotope dilution method was reliable and led to the conclusion that up to their altitudinal limit, legumes may acquire from 59% to more than 90% of their N through symbiotic N-2 fixation depending on the species. These findings were confirmed by the N-15 natural abundance method. Even at the legumes' altitudinal limit all plants investigated showed apparently active nodules. Moreover, a clear host-microsymbiont specificity between plant and rhizobia was evident at high altitudes. This suggests that symbiotic N-2 fixation is well adapted to the climatic and acidic soil conditions in the Alps and contributes, up to the altitudinal limit, a significant amount of N to the N nutrition of legumes. (C) 2000 Elsevier Science Ltd. All rights reserved.