The Bacillus subtilis transcription factor sigma F is a cell-type specific regulatory protein whose activity is governed by SpoIIAB and SpoIIAA and the nucleotides ATP and ADP. SpoIIAB is an anti-sigma factor that binds to sigma F in a manner that is stimulated by ATP, thereby trapping sigma F in an inactive complex. Alternatively, SpoIIAB binds to SpoIIAA in a manner that is stimulated by ADP to form a SpoIIAB.SpoIIAA complex in which SpoIIAB is sequestered from sigma F. SpoIIAB is also a protein kinase that uses ATP to phosphorylate, and thereby inactivate, SpoIIAA. Thus, ATP inhibits sigma F activity both by promoting formation of the SpoIIAB.sigma F complex and by phosphorylation of SpoIIAA. In extension of previous results, we use affinity chromatography to show that SpoIIAB is capable of forming long-lived complexes with sigma F and SpoIIAA and that the formation of these complexes is dependent on ATP and ADP, respectively. Using a DNA template lacking adenosine residues on the non-transcribed strand, we demonstrate that ATP is required for SpoIIAB-mediated inhibition of sigma F-directed RNA synthesis and that this inhibition is prevented by SpoIIAA in a manner that is stimulated by ADP. We show that ADP acts by protecting SpoIIAA from phosphorylation by SpoIIAB and that a mutant protein bearing an amino acid substitution at the site of phosphorylation in SpoIIAA is capable of preventing the inhibition of sigma F in a manner that does not depend on ADP. A principal finding from the investigation is that SpoIIAA restores activity to sigma F that had previously been inhibited by SpoIIAB. This is demonstrated both by the capacity of SpoIIAA to reverse SpoIIAB-mediated inhibition of sigma F-directed RNA synthesis and by its capacity to interact with and disrupt the SpoIIAB. sigma F complex. The results are consistent with a model in which sigma F is controlled by the cellular concentration of unphosphorylated SpoIIAA.