Three distinct fumarases, FumA, FumB and FumC, have been reported in Escherichia coli. While the fumA and fumC gene products are expressed under aerobic cell growth conditions, the FumB fumarase appears to be more abundant during anaerobic growth. To study the transcriptional regulation of the fumB gene, a fumB-lacZ operon fusion was constructed and analyzed in a single copy under a variety of cell culture conditions. Expression of fumB-lacZ was fourfold higher under anaerobic than aerobic growth conditions. This anaerobic response is modulated by the ArcA and Fnr proteins, which function independently as anaerobic activators of fumB gene expression. Cellular iron limitation in a fur mutant caused fumB-lacZ expression to decrease sevenfold while cellular heme limitation decreased fumB gene expression twofold. In addition, fumB-lacZ expression was shown to vary depending on the DNA superhelicity. This study further delineates the regulation of the fumB gene in cell growth.