Pseudomonas fluorescens CHA0 produces hydrogen cyanide (HCN), a secondary metabolite that substantially contributes to this strain's biocontrol ability. Cyanogenesis is induced by oxygen-limiting conditions, but abolished by iron depletion. In P. fluorescens, the anaerobic regulator ANR and the global activator GacA are both required for the maximal expression of the HCN biosynthetic genes hcnABC. The molecular basis of this regulation by ANR and GacA was investigated under conditions of oxygen and iron limitation. A promoter deletion analysis using a translational hcnA'-'lacZ fusion revealed that a conserved FNR/ANR recognition sequence in the -40 promoter region was necessary and sufficient for the regulation by ANR in response to oxygen limitation. Stimulation of hcnA'-'lacZ expression by the addition of iron also depended on the presence of ANR and the FNR/ANR box, but not on GacA, suggesting that in addition to acting as an oxygen-sensitive protein, ANR also responds to iron availability. Expression of the translational hcnA'-'lacZ fusion remained GacA-dependent in hcn promoter mutants that were no longer responsive to ANR, in agreement with earlier evidence for a post-transcriptional regulatory mechanism under GacA control. These data support a model in which cyanogenesis is sequentially activated by ANR at the level of transcription and by components of the GacA network at the level of translation.