Integration host factor protein (IHF) was shown to be required for Fnr- and NarL-dependent activation of the nitrate reductase (narGHJI) operon of Escherichia coli in response to nitrate availability and anaerobiosis. Using a narG-lacZ reporter fusion to evaluate narGHJI expression in vivo both the nitrate and anaerobic dependent controls were severely impaired in a himA mutant compared with the wild type strain. IHF was also required for Fnr-independent anaerobic control of narGHJI expression. In vitro, purified IHF protein was shown to bind to a narG promoter fragment with an apparent dissociation value of 5 nM by use of a gel shift assay. DNase I footprinting studies revealed that IHF protects a 37-base pair region centered 125 base pairs 5' of the narG transcription site. These studies suggest that the IHF protein performs a DNA bending function at the narG promoter to allow nitrate-dependent activation by the NarL regulatory protein, and second, it enhances the Fnr-dependent expression from the narG promoter under anaerobic cell growth conditions. A model whereby three transcriptional activators, NarL, IHF, and Fnr, induce expression of a sigma 70-dependent promoter for the narGHJI operon is discussed.