The sporulation operon spoIIA of Bacillus subtilis consists of three cistrons called spoIIAA, spoIIAB, and spoIIAC. Little is known about the function of spoIIAA and spoIIAB, but spoIIAC encodes a σ factor called σ(F), which is capable of directing the transcription in vitro of genes that are expressed in the forespore chamber of the developing sporangium. We now report that the products of the spoIIA operon constitute a regulatory system in which SpoIIAA is an antagonist of SpoIIAB (or otherwise counteracts the effect of SpoIIAB) and SpoIIAB is, in turn, an antagonist of SpoIIAC (σ(F)). This conclusion is based on the observations that (i) overexpression of spoIIAB inhibits σ(F)-directed gene expression, (ii) a mutation in spoIIAB stimulates σ(F)-directed gene expression, (iii) a mutation in spoIIAA blocks σ(F)-directed gene expression, and (iv) a mutation in spoIIAB relieves the block in σ(F)-directed gene expression caused by a mutation in spoIIAA. The SpoIIAA/SpoIIAB/SpoIIAC regulatory system could play a role in controlling the timing of σ(F)-directed gene expression and/or could be responsible for restricting σ(F)-directed gene expression to the forespore chamber of the sporangium.