Transcriptional repressors and activators are principal control elements in bacterial gene expression. They are involved in the regulation of metabolic pathways, cell division, response to environmental signals, sporulation, replication, to name only a few. Whereas the discovery of these regulators was often fortuitous, a number of molecular, biochemical and biophysical methods have been established that allow to investigate these proteins in great detail and to help understand their functions in the living cell. In this review we focus on a selected set of well characterized transcriptional regulators from Bacillus subtilis and their analysis by methods like EMSA, DNase I footprinting, chemical interference footprinting, in vitro transcription, SELEX, CD measurements, FRET and determination of three-dimensional structure.