The de novo synthesis of NAD in Escherichia coli occurs through the synthesis of quinolinate from aspartate and a 3-carbon compound and its subsequent conversion into NAD, but without the occurrence of nicotinate as a free intermediate. In addition to the de novo pathway, E. coli can also utilize nicotinate or nicotinamide for NAD synthesis by employing a salvage pathway, which is essential for the utilization of exogenous NAD. Several lines of indirect evidence have supported the concept that NAD turnover occurs in various organisms and that the nicotinamide formed can be recycled by the salvage pathway for NAD resynthesis, while other studies have concluded that no significant NAD turnover occurs in E. coli. The present study offers direct evidence for NAD turnover and the presence of a functional pyridine nucleotide cycle in E. coli through crossfeeding experiments and experiments using strains with mutations in the salvage pathway and radioactive nicotinate. The results show that NAD turnover occurs in actively growing nicotinate-auxotroph and prototroph cells of E. coli, which is only evident when the salvage pathway is blocked (resulting in nicotinamide excretion). Conversely, a functional pyridine nucleotide cycle allows normal cells to utilize turnover products for NAD synthesis.