Differential gene expression during sporulation in the prespore and mother cell of Bacillus subtilis is dependent on the correct timing and localization of the activity of specific transcription (sigma) factors. The first sigma factor activated is sigmaF, which directs gene expression specifically in the prespore compartment. Release of sigmaF activity is tightly controlled through a series of complex interactions involving an anti-sigma factor, SpoIIAB, an anti-anti-sigma factor SpoIIAA and a phosphoprotein phosphatase SpoIIE. In vitro studies have shown that SpoIIAB binds to sigmaF, preventing transcription of the sigmaF regulon, and that it can also phosphorylate SpoIIAA, thereby inactivating it. However, non-phosphorylated SpoIIAA can displace sigmaF from SpoIIAB. The SpoIIE phosphatase provides a means of reactivating SpoIIAA-P. We have directly determined the cellular distributions of sigmaF, SpoIIAB, SpoIIAA-P and SpoIIAA during sporulation, using recently developed immunofluorescence methods. While sigmaF activity is restricted to the prespore, the protein is present in both compartments. As development proceeds the sigmaF signal disappears. The anti-sigma factor SpoIIAB is also distributed throughout both cells and rapidly disappears from both cellular compartments soon after sigmaF becomes active. Disappearance of SpoIIAB seems to be closely associated with the activation of the second prespore-specific sigma factor sigmaF. The distribution of phosphorylated SpoIIAA closely mimics that of SpoIIAB, being non-compartmentalized and disappearing soon after sigmaF activation occurs. Significantly, the active, non-phosphorylated form of the anti-anti-sigma factor, SpoIIAA, accumulates in the prespore just before sigmaF becomes active. These results support the hypothesis that the accumulation of SpoIIAA within the prespore is the single most important requirement for activation of sigmaF.