Two-component signal transduction systems enable bacteria to sense, respond, and adapt to a wide range of environments, stressors, and growth conditions. In the prototypical two-component system, a sensor histidine kinase catalyzes its autophosphorylation and then subsequently transfers the phosphoryl group to a response regulator, which can then effect changes in cellular physiology, often by regulating gene expression. The utility of these signaling systems is underscored by their prevalence throughout the bacterial kingdom and by the fact that many bacteria contain dozens, or sometimes hundreds, of these signaling proteins. The presence of so many highly related signaling proteins in individual cells creates both an opportunity and a challenge. Do cells take advantage of the similarity between signaling proteins to integrate signals or diversify responses, and thereby enhance their ability to process information? Conversely, how do cells prevent unwanted cross-talk and maintain the insulation of distinct pathways? Here we address both questions by reviewing the cellular and molecular mechanisms that dictate the specificity of two-component signaling pathways.