A common strategy with two-component systems is to have one protein containing a sensing domain along with the histidine kinase domain. This allows direct regulation of the histidine kinase by the stimulus. A response regulator domain is usually present on a separate protein, and regulates downstream events in the signaling pathway.

The histidine kinase sensors are typically found in the cytoplasmic membrane of the bacterium. This places them in direct contact with the extracellular environment, the sensing domain on the extracellular side of the membrane, the histidine kinase domain on the intracellular side of the membrane. The histidine kinases function as dimers, and in response to the environmental stimulus, each histidine kinase phosphorylates its partner on a conserved histidine residue using ATP as a phospho-donor. This phosphate is then transferred to a conserved aspartic acid residue on the response regulator, the second component in the two-component system. Phosphorylation of the response regulator modulates its ability to mediate downstream signaling in the pathway.

Histidine kinase and response regulator domains can be incorporated into proteins in a variety of ways. Of relevance to the plant two-component systems are the "hybrid" kinases. These contain both histidine kinase and response regulator domains in one protein. In bacteria and yeast, such hybrid kinases participate in phospho-relays: a single phosphate is passed from histidine to aspartate residue on the hybrid kinase, then to a histidine residue on a phospho-relay protein, and from there to an aspartate residue on a separate response regulator.