Asparagine is synthesized from glutamine by the reaction of asparagine synthetase (AS) and is the major nitrogen form in both xylem and phloem sap in rice (Oryza sativa L.). There are two genes encoding AS, OsAS1 and OsAS2, in rice, but the functions of individual AS isoenzymes are largely unknown. Cell type- and NH4(+)-inducible expression of OsAS1 as well as analyses of knockout mutants were carried out in this study to characterize AS1. OsAS1 was mainly expressed in the roots, with in situ hybridization showing that the corresponding mRNA was specifically accumulated in the three cell layers of the root surface (epidermis, exodermis and sclerenchyma) in an NH4(+)-dependent manner. Conversely, OsAS2 mRNA was abundant in leaf blades and sheathes of rice. Although OsAS2 mRNA was detectable in the roots, its content decreased when NH4(+) was supplied. Retrotransposon-mediated knockout mutants lacking AS1 showed slight stimulation of shoot length and slight reduction in root length at the seedling stage. On the other hand, the mutation caused an approximately 80-90% reduction in free asparagine content in both roots and xylem sap. These results suggest that AS1 is responsible for the synthesis of asparagine in rice roots following the supply of NH4(+). Characteristics of the NH4(+)-dependent increase and the root surface cell-specific expression of OsAS1 gene are very similar to our previous results on cytosolic glutamine synthetase1;2 and NADH-glutamate synthase1 in rice roots. Thus, AS1 is apparently coupled with the primary assimilation of NH4(+) in rice roots.