We screened a gene trap library of Arabidopsis thaliana and isolated a line in which a gene encoding a homologue of monofunctional aspartate kinase was trapped by the reporter gene. Aspartate kinase (AK) is a key enzyme in the biosynthsis of aspartate family amino acids such as lysine, threonine, isoleucine, and methionine. In plants, two types of AK are known: one is AK which is sensitive to feedback inhibition by threonine and carries both AK and homoserine dehydrogenase (HSD) activities. The other one is monofunctional, sensitive to lysine and synergistically S-adenosylmethionine, and has only AK activity. We concluded that the trapped gene encoded a monofunctional aspartate kinase and designated as AK-lys3, because it lacked the HSD domain and had an amino acid sequence highly similar to those of the monofunctional aspartate kinases ofA. thaliana. AK-lys3 was highly expressed in xylem of leaves and hypocotyls and stele of roots. Significant expression of this gene was also observed in trichomes after bolting. Slight expression of AK-lys3 was detected in vascular bundles and mesophyll cells of cauline leaves, inflorescence stems, sepals, petals, and stigmas. These results indicated that this aspartate kinase gene was not expressed uniformly but in a spatially specific manner.