Publisher Summary This chapter aims to describe the key experimental techniques used for the analysis of the mechanism of enhancer action. Prokaryotic enhancer share several key properties: activation over a large distance, tight coupling of DNA melting with ATP hydrolysis, high stability of the initiation complexes, and absolute dependence of transcription on the presence of an activator are crucial features of eukaryotic transcription machinery shared only by enhancer-dependent promoters in E. Coli. These properties of prokaryotic enhancers make them a valuable system for the investigation of enhancer function, providing data complementary to in vivo studies of their eukaryotic counterparts. The chapter discusses the progress of studies of the mechanism of prokaryotic enhancer action that is somewhat limited by the lack of an experimental system supporting multiple-round transcription. Some models for the action of eukaryotic enhancers propose existence of a “molecular memory” facilitating enhancer action during multiple-round transcription. This class of the models can only be analyzed properly using a multiple-round transcription assay.