In Escherichia coli, the anaerobic expression of genes encoding the nitrate (narGHJI) and dimethyl sulphoxide (dmsABC) terminal reductases is stimulated by the global anaerobic regulator FNR. The ability of FNR to activate transcription initiation has been proposed to be dependent on protein-protein interactions between RNA polymerase and two activating regions (AR) of FNR, FNR-AR1 and FNR-AR3. To further our understanding of the role of FNR-AR1 and FNR-AR3 in transcription activation, we measured the effects of FNR-AR mutants on expression of the narG and dmsA promoters, PnarG and PdmsA. All the FNR-AR1 (FNR-S73F, FNR-T118A, FNR-S187P), FNR-AR3 (FNR-G85A) and FNR-AR1-AR3 (FNR-G85A-S187P) mutants that were tested decreased expression from PnarG and PdmsA in vivo. Transcription assays of PdmsA also showed that the FNR-AR mutant proteins impaired transcription activation in vitro. Furthermore, DNase I footprinting analysis confirmed that this transcription defect was not a result of altered DNA-binding properties. The function of FNR-S187P and FNR-G85A was also measured in strains containing sigma70 mutants (sigma70-K593A, sigma70-R596A and sigma70-K597A) known to be impaired in FNR-dependent transcription activation. Of all of the combinations analysed, only FNR-G85 and sigma70-K597 showed a genetic interaction, supporting the notion that FNR-AR3 and sigma70 interact functionally in the process of transcription activation. Lastly, the transcription activation defect of the FNR-AR1 and FNR-AR3 mutants was greatly reduced when expression of PnarG was assayed in the presence of nitrate. As these growth conditions promote maximal activity of PnarG as a result of the combined function of NarL, IHF and FNR, these results suggest that the requirements for FNR-AR1 and FNR-AR3 are altered in the presence of additional activators.