Alfalfa (Medicago saliva L.) is a deeply rooted perennial that may be able to remove NO3 from the subsoil beneath the rooting zone of annual crops, Our main objectives were to characterize the potential of two alfalfa cultivars (Agate and Ineffective Agate, an ineffectively nodulated near isoline of Agate) to remove NO3 from the subsoil and to evaluate the influence of available subsoil NO3 on symbiotic N-2 fixation, Low (approximate to 0.3 mM) or high (20 mM) NO3-N concentrations were supplied through a subsoil irrigation system installed in a Hubbard loamy sand soil (sandy, mixed Udorthentic Haploboroll) at Becker, MN, Nitrate uptake and N-2 fixation were evaluated during two regrowth periods using N-15, During fall regrowth in the establishment gear, supply of 20 mM N-15-enriched NO3 through subsoil irrigation did not decrease symbiotic N-2 fixation; during summer regrowth of the first production year, symbiotic N-2 fixation decreased only 40% (from 7.77 to 4.60 g N m(-2)), Even though it had higher yields, Agate alfalfa removed less subsoil NO3-N (1.52 and 5.51 g N m(-2)) than did Ineffective Agate (3.12 and 7.13 g N m(-2)) in each of two harvests where N-15 was applied. Root length and mass after 2 yr were not altered by NO3 treatment or cultivar, This is the first time that direct measurements of subsoil nitrate removal have been made in the field for an N-2-fixing crop, Because it is unlikely that differences in NO3 uptake efficiency could account for such cultivar differences, we propose that assimilation of atmospheric N-2 reduced the capacity of the fixing crop to absorb NO3. These results suggest that non-N-2-fixing alfalfa cultivars would be more useful for bioremediation of nitrate-contaminated sites than standard cultivars.