Bacterial two-component regulatory systems provide a simple stimulus-response coupling mechanism to enable bacteria to detect changes in environmental and intracellular conditions and respond accordingly by adjusting various cellular functions. Since their discovery in the mid-1980s, two-component systems have been the subject of intense investigation. More than a thousand research papers dealing with various aspects of two-component signaling have been published in the Journal of Bacteriology; thus, it felt natural to assemble current studies on the topic into a special issue in the journal. In addition to research articles contributed in response to a call for manuscripts, this special issue includes two minireviews that describe emerging research on two-component systems. One summarizes a broad spectrum of advances from the Bacterial Locomotion and Signal Transduction (BLAST XIV) conference held in January 2017 in New Orleans, LA, and the other focuses on the role of two-component systems in motility and the formation of biofilms. Not surprisingly, both the reviews and original research papers that appear in this issue demonstrate the depth and the breadth of modern research on two-component signal transduction. They showcase studies not only on histidine kinases and response regulators that comprise a two-component system in its classical definition but also on many additional elements that are integrated into or interface with these systems. Best-known examples include chemosensory pathways controlling flagellar motility and other functions that in addition to the CheA/CheY two-component system employ chemoreceptors, adaptors, phosphatases, and modifying enzymes. Functions controlled by two-component systems include phenomena at molecular, cellular, and population levels: gene expression, second-messenger turnover, swimming motility and translocation across surfaces, quorum sensing, biofilm production, host-pathogen as well as beneficial interactions, etc., many of which are documented in this issue. The field has evolved dramatically due to technological advances and interdisciplinary approaches. Studies published in this issue utilized cutting-edge applications in molecular genetics, biochemistry, and structural and cell biology, as well as biophysics and bioinformatics. Thus, we hope that papers published in this issue will be of interest not only to the signal transduction community but also to a much wider readership of the Journal of Bacteriology. We used the journal’s standard procedures for accepting manuscripts for this issue, including thorough peer review of each submission.