Studies were conducted to quantitate the evolution of nitrogen oxides (NO((x))) from soybean [Glycine max (L.) Merr.] leaves during in vivo nitrate reductase (NR) assays with aerobic and anaerobic gas purging. Anaerobic gas purging (N(2) and argon) consistently resulted in greater NO((x)) evolution than did aerobic gas purging (air and O(2)). The evolution of NO((x)) was dependent on gas flow rate and on NO(2) (-) formation in the assay medium; although a threshold level of NO(2) (-) appeared to exist beyond which the rate of NO((x)) evolution did not increase further.The loss of NO((x)) from in vivo NR assays under gas purging explains partially, but not stoichiometrically, the decrease in NO(2) (-) accumulation in in vivo NR assay medium with young soybean leaves. The lack of stoichiometry between NO((x)) evolution and apparent NO(2) (-) loss suggests that other mechanisms are also involved in loss of NO(2) (-) or inhibition of formation of NO(2) (-) during anaerobic and aerobic incubation conditions imposed on the in vivo NR assay of soybean. The mechanism of NO((x)) evolution under the assay conditions imposed and the relevance of this phenomenon to intact plants remains unclear.