This paper reports the identification and functional expression of a gene that is involved in nitrate uptake in plants, a process essential for the assimilation of nitrate and the biological removal of nitrate from the soil solution. The CHL1 gene of Arabidopsis, which when mutated confers resistance to the herbicide chlorate and a decrease in nitrate uptake, was isolated and found to encode a protein with 12 putative membrane-spanning segments. Injection of CHL1 mRNA into Xenopus oocytes produces a nitrate- and pH-dependent membrane depolarization, inward current, and nitrate uptake. These data show that the CHL1 gene encodes an electrogenic nitrate transporter. CHL1 mRNA is found predominantly in roots and displays nitrate- and pH-dependent regulation.