Root NO(3)(-) efflux to the outer medium is a component of NO(3)(-) net uptake and can even overcome influx upon various stresses. Its role and molecular basis are unknown. Following a functional biochemical approach, NAXT1 (for NITRATE EXCRETION TRANSPORTER1) was identified by mass spectrometry in the plasma membrane (PM) of Arabidopsis thaliana suspension cells, a localization confirmed using a NAXT1-Green Fluorescent Protein fusion protein. NAXT1 belongs to a subclass of seven NAXT members from the large NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER family and is mainly expressed in the cortex of mature roots. The passive NO(3)(-) transport activity (K(m) = 5 mM) in isolated root PM, electrically coupled to the ATP-dependant H(+)-pumping activity, is inhibited by anti-NAXT antibodies. In standard culture conditions, NO(3)(-) contents were altered in plants expressing NAXT-interfering RNAs but not in naxt1 mutant plants. Upon acid load, unidirectional root NO(3)(-) efflux markedly increased in wild-type plants, leading to a prolonged NO(3)(-) excretion regime concomitant with a decrease in root NO(3)(-) content. In vivo and in vitro mutant phenotypes revealed that this response is mediated by NAXT1, whose expression is upregulated at the posttranscriptional level. Strong medium acidification generated a similar response. In vitro, the passive efflux of NO(3)(-) (but not of Cl(-)) was strongly impaired in naxt1 mutant PM. This identification of NO(3)(-) efflux transporters at the PM of plant cells opens the way to molecular studies of the physiological role of NO(3)(-) efflux in stressed or unstressed plants.