Expression of the sigF gene encoding a sporulation-specific sigma factor, sigma(F), in Streptomyces aureofaciens is restricted only to sporulation. Gel mobility-shift assays using protein fractions from different developmental stages of S. aureofaciens revealed two different putative proteins specifically bound to the sigF promoter region: a protein (designated RsfA) present in young substrate mycelium, and a protein (designated RsfB) present in the course of sporulation. Based on the characteristic profiles of their appearance during differentiation, RsfA might be a repressor and RsfB an activator of sigF expression. The location of a specific binding site of the repressor-like protein (RsfA) was determined by gel mobility-shift assays of promoter deletion fragments and by DNase I footprinting analysis. The binding site mapped from nucleotides -87 to -25 relative to the transcription start point of the sigF promoter, and overlapped the -35 promoter region. Given the dependence of sigF expression upon whiH, the putative sporulation transcription factor WhiH was overproduced in Escherichia coli and used in the mobility-shift assays with the sigF promoter. However, no specific binding was detected, indicating an indirect dependence of sigF upon whiH.