Two-component system (TCS) signaling circuits regulate numerous cellular processes in response to environmental cues in both prokaryotic and eukaryotic cells. These signaling circuits are all based on phosphoryl-group transfers between histidine and aspartate containing modules of sensor kinase and response regulator proteins. Curiously, the architecture and organization of prokaryotic and eukaryotic two-component systems reveal notable variations, raising the question of whether the input-response specificity that governs the majority of prokaryotic TCSs also governs the eukaryotic ones. In this review, we contrast the TCS architecture and signaling circuits of prokaryotes and eukaryotes, and discuss their possible consequences on signaling specificity.