The two genes, nadA and nadB, responsible for quinolinate biosynthesis from aspartate and dihydroxyacetone phosphate in Escherichia coli were cloned and characterized. Quinolinate (pyridine-2,3-dicarboxylate) is the biosynthetic precursor of the pyridine ring of NAD. Gene nadA was identified by complementation in three different nadA mutant strains. Sequence analysis provided an 840-bp open reading frame coding for a 31,555-Da protein. Gene nadB was identified by complementation in a nadB mutant strain and by the L-aspartate oxidase activity of its gene product. Sequence analysis showed a 1620-bp open reading frame coding for a 60,306-Da protein. For both genes, promoter regions and ribosomal binding sites were assigned by comparison to consensus sequences. The nadB gene product, L-aspartate oxidase, was purified to homogeneity and the N-terminal sequence of 19 amino acids was determined. The enzyme was shown to be specific for L-aspartate. High-copy-number vectors, carrying either gene nadA, nadB or nadA + nadB, increased quinolinate production 1.5-fold, 2.0-fold and 15-fold respectively. Both gene products seem to be equally rate-limiting in quinolinate synthesis.