At the spoIIG promoter phosphorylated Spo0A (Spo0A approximately P) binds 0A boxes overlapping the -35 element, interacting with RNA polymerase to facilitate open complex formation. We have compared in vitro transcription from a series of heteroduplex templates containing denatured regions within the promoters. Transcription from heteroduplex templates with 12, 8, or 6 base pairs denatured was independent of Spo0A approximately P, but heteroduplexes with 4 or 2 base pairs denatured required Spo0A approximately P for maximal levels of transcription. Investigation of the thermal dependence of transcription suggested that strand separation was the primary thermodynamic barrier to transcription initiation but indicated that Spo0A approximately P does not reduce this energetic barrier. Kinetic assays revealed that Spo0A approximately P stimulated both the rate of formation of initiated complexes as well as increasing the number of complexes capable of initiating transcription. These results imply that Spo0A approximately P stimulates transcription at least in part by stabilizing the RNA polymerase-spoIIG complex until contacts between RNA polymerase and the -10 element induce strand separation.