ABSTRACT Compartmentalization of the activities of RNA polymerase sigma factors is a hallmark of formation of spores by Bacillus subtilis . It is initiated soon after the asymmetrically located sporulation division takes place with the activation of σ F in the smaller cell, the prespore. σ F then directs a signal via the membrane protease SpoIIGA to activate σ E in the larger mother cell by processing of pro-σ E . Here, we show that σ E can be activated in the prespore with little effect on sporulation efficiency, implying that complete compartmentalization of σ E activity is not essential for spore formation. σ E activity in the prespore can be obtained by inducing transcription in the prespore of spoIIGA or of sigE *, which encodes a constitutively active form of σ E , but not of spoIIGB , which encodes pro-σ E . We infer that σ E compartmentalization is partially attributed to a competition between the compartments for the activation signaling protein SpoIIR. Normally, SpoIIGA is predominantly located in the mother cell and as a consequence confines σ E activation to it. In addition, we find that CsfB, previously shown to inhibit σ G , is independently inhibiting σ E activity in the prespore. CsfB thus appears to serve a gatekeeper function in blocking the action of two sigma factors in the prespore: it prevents σ G from becoming active before completion of engulfment and helps prevent σ E from becoming active at all.