Anaerobic expression of the focA pfl operon is dependent on the transcription factors ArcA and FNR and transcription is directed by multiple, anaerobically regulated promoters. A FNR-binding site is centred at -41.5 bp relative to the P6 promoter, inactivation of which severely impairs anaerobic expression of the complete operon. Mutations were introduced into this binding site to create a consensus recognition site for the cAMP-receptor protein, CRP (CC-site), and one that was recognised by both CRP and FNR (CF-site). Transcription directed by these mutant binding sites in vivo in different promoter constructs was analysed by primer extension and by constructing lacZ operon fusions. With a derivative including only the P6 promoter and the CF-binding site, transcription was shown to be independent of oxygen and was activated by CRP or FNR. In agreement with previous findings, FNR only activated transcription anaerobically. In a construct including the CC-binding site transcription was strong. CRP dependent and initiated at the identical site to the wild-type promoter. Transcription activation from the CC-site was exquisitely sensitive to low cAMP concentration. Surprisingly, in a crp mutant, anaerobically inducible, FNR-dependent transcription directed by the CC-site was detected, indicating that FNR can recognise a consensus CRP-binding site in vivo. A strain unable to synthesise CRP or FNR exhibited no transcription from the P6 promoter. Essentially the same results were observed in a series of constructs that also included the promoter P7 and its regulatory sequences. Evidence is also presented which demonstrates that CRP activates transcription from the natural FNR-binding site of the P6 promoter. In vitro DNA-binding studies showed that CRP specifically interacted with the FNR-binding site, protecting exactly the same sequence as that protected by the FNR protein. Interaction of CRP with the natural FNR-binding site was reduced greater than 50-fold compared to its interaction with the mutant CC-binding site. Although we could not demonstrate that FNR interacted with the CC-binding site in vitro, it did bind to the CF-site giving the same protection as observed with the wild-type FNR-binding site. FNR also activated transcription from the CF-site in vitro, giving further support to the idea that a single functional DNA half-site is sufficient to direct binding and transcription activation by a dimeric transcription factor.