Temporally and environmentally regulated gene expression in prokaryotes occurs primarily at the level of transcription initiation. Two main modes of regulation have been described, including either the binding of a repressor that blocks transcription or the interaction of a positive regulator with the transcription complex, leading to transcription initiation. Several classes can be distinguished among positive regulators according to their mechanisms of action. This review describes the different types of positive regulators identified in Bacillus subtilis, a gram-positive bacterium. These include accessory regulatory polypeptides, classical positive regulators that bind to target sites located just upstream from the promoter, ambiactive regulators that can act both positively and negatively, antiterminators, two-component signal transduction systems, and positive regulators associated with specific secondary sigma factors.