L-Aspartate oxidase is a monomeric flavoprotein that catalyzes the first step in the de novo biosynthetic pathway for pyridine nucleotide formation under both aerobic and anaerobic conditions. In spite of the physiological importance of this biosynthesis in particular in facultative aerobic organisms, such as Escherichia coli, little is known about the electron acceptor of reduced L-aspartate oxidase in the absence of oxygen. In this report, evidence is presented which suggests that in vitro fumarate can play such a role. L-Aspartate oxidase binds succinate and fumarate with Kd values of 0.24 mM and 0.22 mM, respectively. A competitive behaviour was observed for these two dicarboxylic acids towards iminoaspartate and sulfite ions. Photoreduction experiments suggest that fumarate and succinate bind at or close to the active site of the molecule. A new fumarate reductase activity of L-aspartate oxidase is reported using benzylviologen or L-aspartate as reductants and fumarate as oxidant. Steady-state kinetics for the oxidase and the fumarate reductase activity of L-aspartate oxidase were obtained using either fumarate or oxygen as electron acceptor and L-aspartate as electron donor. Finally, succinate was identified as the product of the L-aspartate:fumarate oxidoreductase activity using radiolabeled fumarate under anaerobic conditions. The results suggest that fumarate can be a valuable alternative to oxygen as a substrate for L-aspartate oxidase.