During endospore formation in Bacillus subtilis an asymmetric division produces two cells, forespore and mother cell, which follow different developmental paths. Commitment to the forespore-specific developmental path is controlled in part by the activation of the forespore-specific RNA polymerase sigma factor, sigma F. Activity of sigma F is inhibited in the mother cell by the anti-sigma factor SpoIIAB. In the forespore, sigma F directs transcription of the structural gene for sigma G. However, sigma G does not become active until after engulfment of the forespore is complete. This sigma G activity is dependent upon the products of the spoIIIA operon. We showed that sigma G is present but mostly inactive in a spoIIIA mutant. We also demonstrated that the anti-sigma factor SpoIIAB can bind to sigma G in vitro. Moreover, a mutant form of sigma G that binds SpoIIAB inefficiently in vitro was shown to function independently of SpoIIIA during sporulation. These and previously reported results support a model in which SpoIIAB functions as an inhibitor of sigma G activity during sporulation. Therefore, we propose that the anti-sigma factor SpoIIAB antagonizes both sigma F and sigma G activities, and that this antagonism is relieved in the forespore in two stages. In the first stage, which follows septation, a SpoIIAA-dependent mechanism partially relieves SpoIIAB inhibition of sigma F activity in the forespore. In the second stage, which follows forespore engulfment, a SpoIIIA-dependent process inactivates SpoIIAB in the forespore, resulting in the activation of sigma G.