The sigma subunit of RNA polymerase is a critical factor in positive control of transcription initiation. Primary sigma factors are essential proteins required for vegetative growth, whereas alternative sigma factors mediate transcription in response to various stimuli. Late gene expression during flagellum biosynthesis in Salmonella typhimurium is dependent upon an alternative sigma factor, sigma28, the product of the fliA gene. We have characterized the intermediate complexes formed by sigma28 holoenzyme on the pathway to open complex formation. Interactions with the promoter for the flagellin gene fliC were analyzed using DNase I and KMnO4 footprinting over a range of temperatures. We propose a model in which closed complexes are established in the upstream region of the promoter, including the -35 element, but with little significant contact in the -10 element or downstream regions of the promoter. An isomerization event extends the DNA contacts into the -10 element and the start site, with loss of the most distal upstream contacts accompanied by DNA melting to form open complexes. Melting occurs efficiently even at 16 degrees C. Once open complexes have formed, they are unstable to heparin challenge even in the presence of nucleoside triphosphates, which have been observed to stabilize open complexes at rRNA promoters.