Electron microscopic examination of sporulating cultures of wild-type Bacillus subtilis revealed that the morphological events previously characterized as stages II and III can be divided into four substages, namely, stages IIi, IIii, IIiii, and III. The ultrastructural phenotypes of several stage II mutant strains indicate that each of the four substages has a biochemical and genetic basis. Two of the genes needed for the transition from stage II to stage III encode transcription factors sigma E and sigma F. Their roles during spore morphogenesis have been the subject of much speculation. We now show that sigma E controls genes involved in the morphological transition from stage IIi to stage IIii and then stage IIiii, while the transition to stage III may be determined by genes controlled by sigma F. The results also indicate the existence of at least two undiscovered sporulation genes involved in B. subtilis spore morphogenesis.