Arginine is an essential amino acid necessary for protein synthesis and is also a nitrogen storage compound. The genes encoding the enzymes of arginine biosynthesis in plants are not well characterized and have mainly been predicted from homologies to bacterial and fungal genes. We report the cloning and characterization of the TUMOR PRONE5 (TUP5) gene of Arabidopsis (Arabidopsis thaliana) encoding an acetylornithine aminotransferase (ACOAT), catalyzing the fourth step of arginine biosynthesis. The free arginine content was strongly reduced in the chemically induced recessive mutant tup5-1, root growth was restored by supplementation with arginine and its metabolic precursors, and a yeast (Saccharomyces cerevisiae) ACOAT mutant was complemented by TUP5. Two null alleles of TUP5 caused a reduced viability of gametes and embryo lethality, possibly caused by insufficient Arg supply from maternal tissue. TUP5 expression is positively regulated by light, and a TUP5-green fluorescent protein was localized in chloroplasts. tup5-1 has a unique light-dependent short root phenotype. Roots of light-grown tup5-1 seedlings switch from indeterminate growth to determinate growth with arresting cell production and an exhausted root apical meristem. The inhibitory activity was specific for blue light, and the inhibiting light was perceived by the root. Thus, tup5-1 reveals a novel role of amino acids and blue light in regulating root meristem function.