Bacteria, fungi, and plants exploit histidine sensor kinase/response regulators to mobilize complex responses to inputs as diverse as environmental stimuli and hormonal regulation. More than 50 such two-component systems are found in many organisms, yet the mechanisms of signal perception, phosphotransfer regulation, and even the nature of the activating signals remain poorly defined. Here we resolve each phosphate transfer event in vivo for the Agrobacterium tumefaciens virulence two-component system VirA/VirG. The input signals for this system are known, and the complex autocatalytic regulation of the signaling components has been removed. Two separate and independent phosphotransfer events are resolved, an initial ATP-->sensorHis approximately PO(4)-->receiver approximately PO(4), that may be activated by xenognostic sugar/low pH, and a subsequent ATP-->His approximately PO(4)-->VirG approximately PO(4) that requires xenognostic phenol activation. The identification of these separate pathways places biochemical limits on the regulated steps in this two-component signal transduction module and further extends the model of how a single sensor is able to integrate multiple input stimuli.