The use of nitrogen (N) fertilizers has created international concern as denitrification of fertilizer-derived nitrate could result in higher rates of N2O release to the atmosphere. The discovery that many eukaryotic fungi can transform N through nitrification and denitrification pathways is significant from both biochemical and ecological perspectives, because fungi dominate microbial metabolism in many terrestrial ecosystems. It is known that roots from semi-arid grasslands are colonized by a diverse fungal community, and suppression of such fungal biomass could cause a substantial reduction of N2O production. N2O production by sixteen root-associated soil fungal species was estimated by analyzing gas samples from pure cultures isolated from blue-grama rhizosphere soils at the Sevilleta Long-Term Ecological Research site in Central New Mexico. The results demonstrate that N2O production is common among fungi from this arid grassland. In addition, results demonstrate the value of a Tunable- Diode-Laser (TDL) approach for measuring N2O production by fungi in pure culture.