Nitrous oxide (N(2)O) is a key atmospheric greenhouse gas that contributes to global climatic change through radiative warming and depletion of stratospheric ozone. In this report, N(2)O flux was monitored simultaneously with photosynthetic CO(2) and O(2) exchanges from intact canopies of 12 wheat seedlings. The rates of N(2)O-N emitted ranged from <2 pmol x m(-2) x s(-1) when NH(4)(+) was the N source, to 25.6 +/- 1.7 pmol x m(-2) x s(-1) (mean +/- SE, n = 13) when the N source was shifted to NO(3)(-). Such fluxes are among the smallest reported for any trace gas emitted by a higher plant. Leaf N(2)O emissions were correlated with leaf nitrate assimilation activity, as measured by using the assimilation quotient, the ratio of CO(2) assimilated to O(2) evolved. (15)N isotopic signatures on N(2)O emitted from leaves supported direct N(2)O production by plant NO(3)(-) assimilation and not N(2)O produced by microorganisms on root surfaces and emitted in the transpiration stream. In vitro production of N(2)O by both intact chloroplasts and nitrite reductase, but not by nitrate reductase, indicated that N(2)O produced by leaves occurred during photoassimilation of NO(2)(-) in the chloroplast. Given the large quantities of NO(3)(-) assimilated by plants in the terrestrial biosphere, these observations suggest that formation of N(2)O during NO(2)(-) photoassimilation could be an important global biogenic N(2)O source.