NAD is a ubiquitous coenzyme involved in oxidation-reduction reactions and is synthesized by way of quinolinate. Animals and some bacteria synthesize quinolinate from tryptophan, whereas other bacteria synthesize quinolinate from aspartate (Asp) using l-Asp oxidase and quinolinate synthase. We show here that Arabidopsis (Arabidopsis thaliana) uses the Asp-to-quinolinate pathway. The Arabidopsis l-Asp oxidase or quinolinate synthase gene complemented the Escherichia coli mutant defective in the corresponding gene, and T-DNA-based disruption of either of these genes, as well as of the gene coding for the enzyme quinolinate phosphoribosyltransferase, was embryo lethal. An analysis of functional green fluorescent protein-fused constructs and in vitro assays of uptake into isolated chloroplasts demonstrated that these three enzymes are located in the plastid.