We have determined the nucleotide sequence of the promoter for aB. subtilis gene (the 0.4 kb gene) whose transcription is under developmental control. Transcription of the 0.4 kb gene is turned on at the onset of sporulation; this RNA synthesis depends on the products of the B. subtilis regulatory genes (the spoO loci) that control the initiation of development. Recognition of the 0.4 kb gene promoter in vitro is dictated by novel species of B. subtilis RNA polymerase sigma factor known as sigma 37 and sigma 29 but not by the principal B. subtilis sigma factor sigma 55. Using S1 nuclease mapping, runoff transcription and dinucleotide priming, we have identified dual startpoints (separated by about 10 bp) for sigma37-directed transcription of the 0.4 kb gene These start-points correspond closely to the 5' termini of 0.4 kb RNA synthesized in vivo during the course of sporulation. Two forms of sigma37 containing RNA polymerase were distinguished that preferentially utilize either the upstream or the downstream startpoint in vitro. We investigated the requirements for sigma37-directed transcription by constructing in vitro deletion mutations that extend from the upstream direction into the 0.4 kb promoter region. One such deletion, which terminates 40 and 51 bp upstream from the transcription startpoints (thereby removing a highly AT-rich 26 bp sequence), completely eliminates transcription from the downstream startpoint but only partially inhibits transcription from the upstream startpoint. A second deletion that terminates at the upstream startpoint completely prevents transcription from both initiation sites. The implications of 0.4 kb gene promoter structure for developmentally regulated transcription of B, subtilis are discussed.