Compartmentalized gene expression during sporulation is initiated after asymmetric division by cell-specific activation of the transcription factors σF and σE. Synthesis of these σ factors, and their regulatory proteins, requires the activation (phosphorylation) of Spo0A by the phosphorelay signalling system. We report here a novel regulatory function of the anti-anti-σF SpoIIAA as inhibitor of Spo0A activation. This effect did not require σF activity, and it was abolished by expression of the phosphorelay-independent form Spo0A-Sad67 indicating that SpoIIAA directly interfered with Spo0A∼P generation. IPTG-directed synthesis of the SpoIIE phosphatase in a strain carrying a multicopy plasmid coding for SpoIIAA and its specific inhibitory kinase SpoIIAB blocked Spo0A activation suggesting that the active form of the inhibitor was SpoIIAA and not SpoIIAA-P. Furthermore, expression of the non-phosphorylatable mutant SpoIIAAS58A (SpoIIAA-like), but not SpoIIAAS58D (SpoIIAA-P-like), completely blocked Spo0A-dependent gene expression. Importantly, SpoIIAA expressed from the chromosome under the control of its normal spoIIA promoter showed the same negative effect regulated not only by SpoIIAB and SpoIIE but also by septum morphogenesis. These findings are discussed in relation to the potential contribution of this novel inhibitory feedback with the proper activation of σF and σE during development.