Bacillus subtilis r D is an alternative r factor that possesses a core-independent promoter À10 element binding specificity despite the lack of a distinct footprint on its cognate promoter. We wished to determine whether this property is common to alternative r factors. To this end, we overexpressed B. subtilis r B in Escherichia coli and analyzed its DNA binding ability by electrophoretic mobility shift assay and DNase I footprinting. The major complex formed by r B and its cognate promoter DNA is heparin-sensitive. However, in contrast to the À10 element binding specificity observed for B. subtilis r D , the promoter binding of r B is specific for the À35 element. These and other results clearly demonstrate that alternative r factors possess different promoter-binding characteristics, and make coreindependent contributions to recognition of their cognate promoters.