The Arabidopsis CHL1 (AtNRT1) gene confers sensitivity to the herbicide chlorate and encodes a nitrate-regulated nitrate transporter. However, how CHL1 participates in nitrate uptake in plants is not yet clear. In this study, we examined the in vivo function of CHL1 with in vivo uptake measurements and in situ hybridization experiments. Under most conditions tested, the amount of nitrate uptake by a chl1 deletion mutant was found to be significantly less than that of the wild type. This uptake deficiency was reversed when a CHL1 cDNA clone driven by the cauliflower mosaic virus 35S promoter was expressed in transgenic chl1 plants. Furthermore, tissue-specific expression patterns showed that near the root tip, CHL1 mRNA is found primarily in the epidermis, but further from the root tip, the mRNA is found in the cortex or endodermis. These results are consistent with the involvement of CHL1 in nitrate uptake at different stages of root cell development. A functional analysis in Xenopus oocytes indicated that CHL1 is a low-affinity nitrate transporter with a K(m) value of approximately 8.5 mM for nitrate. This finding is consistent with the chlorate resistance phenotype of chl1 mutants. However, these results do not fit the current model of a single, constitutive component for the low-affinity uptake system. To reconcile this discrepancy and the complex uptake behavior observed, we propose a "two-gene" model for the low-affinity nitrate uptake system of Arabidopsis.